Methods and systems of using exosomes for determining phenotypes

ABSTRACT

Exosomes can be used for detecting biomarkers for diagnostic, therapy-related or prognostic methods to identify phenotypes, such as a condition or disease, for example, the stage or progression of a disease. Cell-of-origin exosomes can be used in profiling of physiological states or determining phenotypes. Biomarkers or markers from cell-of-origin specific exosomes can be used to determine treatment regimens for diseases, conditions, disease stages, and stages of a condition, and can also be used to determine treatment efficacy. Markers from cell-of-origin specific exosomes can also be used to identify conditions of diseases of unknown origin.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.12/658,452, filed Feb. 5, 2010, which is a continuation of U.S. patentapplication Ser. No. 12/591,226 filed Nov. 12, 2009, which claims thebenefit of U.S. Provisional Application Nos. 61/114,045, filed Nov. 12,2008; 61/114,058, filed Nov. 12, 2008; 61/114,065, filed Nov. 13, 2008;61/151,183, filed Feb. 9, 2009; 61/278,049, filed Oct. 2, 2009;61/250,454, filed Oct. 9, 2009; and 61/253,027 filed Oct. 19, 2009. Thisapplication is also related to Customer Number 96600, Docket Number37901-706.302, filed herewith on Jan. 19, 2011, and U.S. applicationSer. No. 12/609,847, filed Oct. 30, 2009, which claims the benefit ofU.S. Provisional Application Nos. 61/109,742, filed Oct. 30, 2008;61/112,571, filed Nov. 7, 2008, as well as the provisional applicationsreferenced with respect to U.S. application Ser. No. 12/591,226. Theentire content of these applications is hereby incorporated byreference.

BACKGROUND

A critical need for disease detection, prognostic prediction,monitoring, and therapeutic decisions is improved assay sensitivity andspecificity. At present, biomarkers (proteins, peptides, lipids, RNAs,DNA and modifications thereof for disease-associated molecularalterations) for conditions and diseases, such as cancer, rely almostexclusively on obtaining samples from tissue to identify the conditionor disease. Methods to obtain these tissues of interest for analysis areoften invasive, costly and pose complication risks for the patient.Furthermore, use of bodily fluids to isolate or detect biomarkers oftensignificantly dilutes a biomarker resulting in readouts that lackrequisite sensitivity. Additionally, most biomarkers are produced in lowor moderate amounts in normal tissues other than the diseased tissue andthus this lack of specificity can also be problematic.

The identification of specific biomarkers, such as DNA, RNA and proteinscan provide bio-signatures that are used for the diagnosis, prognosis,or theranosis of a condition or disease. Exosomes are a good source forassessing one or more biomarkers that are present in or on the surfaceof an exosome. Furthermore, identifying particular characteristics of anexosome (e.g., size, surface antigens, cell-of-origin) can itselfprovide a diagnostic, prognostic or theranostic readout.

The secretion of exosomes by cancerous cells, other diseased cells, orat certain times of a physiological process (e.g., pregnancy), can beleveraged to aid in diagnosis as well as individualized treatmentdecisions. Exosomes have been found in a number of body fluids,including blood plasma, breast milk, bronchoalveolar lavage fluid andurine. Exosomes also take part in the communication between cells, astransport vehicles for proteins, RNAs, DNAs, viruses, and prions.

The present inventions provide an improvement to prior art assays.Products and process are provided for improved assay sensitivity andspecificity, allowing for disease detection, prognostic prediction,disease monitoring, disease staging, and therapeutic decision-making, aswell as physiological state identification. Products and processesinclude cell-of-origin specific selection of exosomes and analysis oftheir protein composition, RNA composition, DNA composition, lipidprofile, and relevant metabolic and/or epigenetic modifications of theseanalytes. Also provided herein are methods of determining biomarkers andbio-signatures for exosomes without prior concentration or purificationof the exosomes from a sample.

SUMMARY

Disclosed herein are methods and compositions for characterizing aphenotype by analyzing an exosome. Characterizing a phenotype for asubject or individual may include, but is not limited to, the diagnosisof a disease or condition, the prognosis of a disease or condition, thedetermination of a disease stage or a condition stage, a drug efficacy,a physiological condition, organ distress or organ rejection, disease orcondition progression, therapy-related association to a disease orcondition, or a specific physiological or biological state.

The method can include determining a bio-signature of an exosome in abiological sample from a subject and characterizing a phenotype in saidsubject based on the bio-signature. Characterizing can also be based ondetermining the amount of exosomes in a biological sample. Thecharacterization of the phenotype can be performed with at least 70, 80or 90% sensitivity, specificity, or both.

The exosome can be isolated or concentrated prior to determining anexosomal bio-signature. The bio-signature can comprise an expressionlevel, presence, absence, mutation, copy number variation, truncation,duplication, insertion, modification, sequence variation, or molecularassociation of a biomarker. The bio-signature can also comprisequantification of isolated exosomes, temporal evaluation of thevariation in exosomal half-life, circulating exosomal half-life,exosomal metabolic half-life, or the activity of an exosome.

The exosome can be a cell-of-origin specific exosome. The exosome can bederived from a tumor or cancer cell. The cell-of-origin for an exosomecan be a lung, pancreas, stomach, intestine, bladder, kidney, ovary,testis, skin, colorectal, breast, prostate, brain, esophagus, liver,placenta, or fetal cell.

One or more biomarkers of an exosome can be assessed for characterizinga phenotype. The biomarker can be a nucleic acid, peptide, protein,lipid, antigen, carbohydrate or proteoglycan, such as DNA or RNA. TheRNA can be mRNA, miRNA, snoRNA, snRNA, rRNAs, tRNAs, siRNA, hnRNA, orshRNA. The biomarker can be an antigen selected from FIG. 1, or abiomarker selected from a table listed in FIG. 3-60. One or morebiomarkers can be assessed and used to characterize a phenotype. Thebio-signature can comprise one or more miRNAs selected from the groupconsisting of: miR-9, miR-629, miR-141, miR-671-3p, miR-491, miR-182,miR-125a-3p, miR-324-5p, miR-148b, and miR-222. The bio-signature can beused to characterize a phenotype, such as prostate cancer. Otherbiomarkers can be selected from the group consisting of: CD9, PSCA(prostate stem cell antigen), TNFR, CD63, MFG-E8, EpCam, Rab, CD81,STEAP, PCSA (prostate cell surface antigen), PSM (or PSMA, prostatespecific membrane antigen), 5T4, CD59, CD66, CD24 and B7H3. Detecting aplurality of biomarkers can provide greater sensitivity or specificityas compared to detecting less than a plurality of biomarkers.

Methods of multiplexing, or multiplex analysis of, a plurality ofexosomes are also provided. Multiplexing a plurality of exosomes cancomprise applying said plurality of exosomes to a plurality ofparticles, wherein each particle of a subset of the plurality ofparticles is coupled to a different capture agent, capturing a subset ofsaid plurality of exosomes; and, detecting one or more biomarkers of thecaptured exosomes. Multiplexing can also be performed using an array,wherein the capture agents are attached to an array instead of particlesor beads.

Also provided herein are isolated exosomes. The isolated exosome cancomprise any one or more biomarkers disclosed herein, such as a specificcombination of biomarkers. Compositions comprising the one or moreisolated exosomes are also provided. The composition can comprise asubstantially enriched population of exosomes. The population ofexosomes can be substantially homogeneous for one or more specificbiomarkers, for a particular bio-signature, or derived from a specificcell type.

Detection systems, microfluidic devices, and kits for assessing one ormore exosomes, such as for the isolation, separation, or detection ofone or more exosomes, are also provided.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (a)-(g) represents a table which lists exemplary cancers bylineage, group comparisons of cells/tissue, and specific disease statesand antigens specific to those cancers, group cell/tissue comparisonsand specific disease states. Furthermore, the antigen can be abiomarker. The one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 2 (a)-(f) represents a table which lists exemplary cancers bylineage, group comparisons of cells/tissue, and specific disease statesand binding agents specific to those cancers, group cell/tissuecomparisons and specific disease states.

FIG. 3 (a)-(b) represents a table which lists exemplary breast cancerbiomarkers that can be derived and analyzed from exosomes specific tobreast cancer to create a breast cancer specific exosome bio-signature.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 4 (a)-(b) represents a table which lists exemplary ovarian cancerbiomarkers that can be derived from and analyzed from exosomes specificto ovarian cancer to create an ovarian cancer specific exosomebio-signature. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 5 represents a table which lists exemplary lung cancer biomarkersthat can be derived from and analyzed from exosomes specific to lungcancer to create a lung cancer specific exosome bio-signature.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 6 (a)-(d) represents a table which lists exemplary colon cancerbiomarkers that can be derived from and analyzed from exosomes specificto colon cancer to create a colon cancer specific exosome bio-signature.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 7 represents a table which lists exemplary biomarkers specific toan adenoma versus a hyperplastic polyp that can be derived and analyzedfrom exosomes specific to adenomas versus hyperplastic polyps.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 8 is a table which lists exemplary biomarkers specific toinflammatory bowel disease (IBD) versus normal tissue that can bederived and analyzed from exosomes specific to inflammatory boweldisease versus normal tissue. Furthermore, the one or more biomarkerscan be present or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 9( a)-(c) represents a table which lists exemplary biomarkersspecific to an adenoma versus colorectal cancer (CRC) that can bederived and analyzed from exosomes specific to adenomas versuscolorectal cancer. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 10 represents a table which lists exemplary biomarkers specific toIBD versus CRC that can be derived and analyzed from exosomes specificto IBD versus CRC. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 11 (a)-(b) represents a table which lists exemplary biomarkersspecific to CRC Dukes B versus Dukes C-D that can be derived andanalyzed from exosomes specific to CRC Dukes B versus Dukes C-D.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 12( a)-(d) represents a table which lists exemplary biomarkersspecific to an adenoma with low grade dysplasia versus an adenoma withhigh grade dysplasia that can be derived and analyzed from exosomesspecific to an adenoma with low grade dysplasia versus an adenoma withhigh grade dysplasia. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 13( a)-(b) represents a table which lists exemplary biomarkersspecific to ulcerative colitis (UC) versus Crohn's Disease (CD) that canbe derived and analyzed from exosomes specific to UC versus CD.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 14 represents a table which lists exemplary biomarkers specific toa hyperplastic polyp versus normal tissue that can be derived andanalyzed from exosomes specific to a hyperplastic polyp versus normaltissue. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 15 is a table which lists exemplary biomarkers specific to anadenoma with low grade dysplasia versus normal tissue that can bederived and analyzed from exosomes specific to an adenoma with low gradedysplasia versus normal tissue. Furthermore, the one or more biomarkerscan be present or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 16 is a table which lists exemplary biomarkers specific to anadenoma versus normal tissue that can be derived and analyzed fromexosomes specific to an adenoma versus normal tissue. Furthermore, theone or more biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 17 represents a table which lists exemplary biomarkers specific toCRC versus normal tissue that can be derived and analyzed from exosomesspecific to CRC versus normal tissue. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 18 is a table which lists exemplary biomarkers specific to benignprostatic hyperplasia that can be derived from and analyzed fromexosomes specific to benign prostatic hyperplasia. Furthermore, the oneor more biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 19( a)-(c) represents a table which lists exemplary prostate cancerbiomarkers that can be derived from and analyzed from exosomes specificto prostate cancer to create a prostate cancer specific exosomebio-signature. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 20( a)-(c) represents a table which lists exemplary melanomabiomarkers that can be derived from and analyzed from exosomes specificto melanoma to create a melanoma specific exosome bio-signature.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 21( a)-(b) represents a table which lists exemplary pancreaticcancer biomarkers that can be derived from and analyzed from exosomesspecific to pancreatic cancer to create a pancreatic cancer specificexosome bio-signature. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 22 is a table which lists exemplary biomarkers specific to braincancer that can be derived from and analyzed from exosomes specific tobrain cancer to create a brain cancer specific exosome bio-signature.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 23( a)-(b) represents a table which lists exemplary psoriasisbiomarkers that can be derived from and analyzed from exosomes specificto psoriasis to create a psoriasis specific exosome bio-signature.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 24( a)-(c) represents a table which lists exemplary cardiovasculardisease biomarkers that can be derived from and analyzed from exosomesspecific to cardiovascular disease to create a cardiovascular diseasespecific exosome bio-signature. Furthermore, the one or more biomarkerscan be present or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 25 is a table which lists exemplary biomarkers specific tohematological malignancies that can be derived from and analyzed fromexosomes specific to hematological malignancies to create a specificexosome bio-signature for hematological malignancies. Furthermore, theone or more biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 26( a)-(b) represents a table which lists exemplary biomarkersspecific to B-Cell Chronic Lymphocytic Leukemias that can be derivedfrom and analyzed from exosomes specific to B-Cell Chronic LymphocyticLeukemias to create a specific exosome bio-signature for B-Cell ChronicLymphocytic Leukemias. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 27 is a table which lists exemplary biomarkers specific to B-CellLymphoma and B-Cell Lymphoma-DLBCL that can be derived from and analyzedfrom exosomes specific to B-Cell Lymphoma and B-Cell Lymphoma-DLBCL.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 28 represents a table which lists exemplary biomarkers specific toB-Cell Lymphoma-DLBCL-germinal center-like and B-CellLymphoma-DLBCL-activated B-cell-like and B-cell lymphoma-DLBCL that canbe derived from and analyzed from exosomes specific to B-CellLymphoma-DLBCL-germinal center-like and B-Cell Lymphoma-DLBCL-activatedB-cell-like and B-cell lymphoma-DLBCL. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 29 represents a table which lists exemplary Burkitt's lymphomabiomarkers that can be derived from and analyzed from exosomes specificto Burkitt's lymphoma to create a Burkitt's lymphoma specific exosomebio-signature. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 30( a)-(b) represents a table which lists exemplary hepatocellularcarcinoma biomarkers that can be derived from and analyzed from exosomesspecific to hepatocellular carcinoma to create a specific exosomebio-signature for hepatocellular carcinoma. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 31 is a table which lists exemplary biomarkers for cervical cancerthat can be derived from and analyzed from exosomes specific to cervicalcancer. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 32 represents a table which lists exemplary biomarkers forendometrial cancer that can be derived from and analyzed from exosomesspecific to endometrial cancer to create a specific exosomebio-signature for endometrial cancer. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 33( a)-(b) represents a table which lists exemplary biomarkers forhead and neck cancer that can be derived from and analyzed from exosomesspecific to head and neck cancer to create a specific exosomebio-signature for head and neck cancer. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 34 represents a table which lists exemplary biomarkers forinflammatory bowel disease (IBD) that can be derived from and analyzedfrom exosomes specific to IBD to create a specific exosome bio-signaturefor IBD. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 35 is a table which lists exemplary biomarkers for diabetes thatcan be derived from and analyzed from exosomes specific to diabetes tocreate a specific exosome bio-signature for diabetes. Furthermore, theone or more biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 36 is a table which lists exemplary biomarkers for Barrett'sEsophagus that can be derived from and analyzed from exosomes specificto Barrett's Esophagus to create a specific exosome bio-signature forBarrett's Esophagus. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 37 is a table which lists exemplary biomarkers for fibromyalgiathat can be derived from and analyzed from exosomes specific tofibromyalgia. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 38 represents a table which lists exemplary biomarkers for strokethat can be derived from and analyzed from exosomes specific to stroketo create a specific exosome bio-signature for stroke. Furthermore, theone or more biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 39 is a table which lists exemplary biomarkers for MultipleSclerosis (MS) that can be derived from and analyzed from exosomesspecific to MS to create a specific exosome bio-signature for MS.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 40( a)-(b) represents a table which lists exemplary biomarkers forParkinson's Disease that can be derived from and analyzed from exosomesspecific to Parkinson's Disease to create a specific exosomebio-signature for Parkinson's Disease. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 41 represents a table which lists exemplary biomarkers forRheumatic Disease that can be derived from and analyzed from exosomesspecific to Rheumatic Disease to create a specific exosome bio-signaturefor Rheumatic Disease. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 42( a)-(b) represents a table which lists exemplary biomarkers forAlzheimers Disease that can be derived from and analyzed from exosomesspecific to Alzheimers Disease to create a specific exosomebio-signature for Alzheimers Disease. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 43 is a table which lists exemplary biomarkers for Prion Diseasesthat can be derived from and analyzed from exosomes specific to PrionDiseases to create a specific exosome bio-signature for Prion Diseases.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 44 represents a table which lists exemplary biomarkers for sepsisthat can be derived from and analyzed from exosomes specific to sepsisto create a specific exosome bio-signature for sepsis. Furthermore, theone or more biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 45 is a table which lists exemplary biomarkers for chronicneuropathic pain that can be derived from and analyzed from exosomesspecific to chronic neuropathic pain. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 46 is a table which lists exemplary biomarkers for peripheralneuropathic pain that can be derived from and analyzed from exosomesspecific to peripheral neuropathic pain. Furthermore, the one or morebiomarkers can be present or absent, underexpressed or overexpressed,mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 47 represents a table which lists exemplary biomarkers forSchizophrenia that can be derived from and analyzed from exosomesspecific to Schizophrenia to create a specific exosome bio-signature forSchizophrenia. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 48 is a table which lists exemplary biomarkers for bipolar disorderor disease that can be derived from and analyzed from exosomes specificto bipolar disorder to create a specific exosome bio-signature forbipolar disorder. Furthermore, the one or more biomarkers can be presentor absent, underexpressed or overexpressed, mutated, or modified, suchas epigentically modified or post-translationally modified.

FIG. 49 is a table which lists exemplary biomarkers for depression thatcan be derived from and analyzed from exosomes specific to depression tocreate a specific exosome bio-signature for depression. Furthermore, theone or more biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 50 is a table which lists exemplary biomarkers for gastrointestinalstromal tumor (GIST) that can be derived from and analyzed from exosomesspecific to GIST to create a specific exosome bio-signature for GIST.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 51( a)-(b) represent sa table which lists exemplary biomarkers forrenal cell carcinoma (RCC) that can be derived from and analyzed fromexosomes specific to RCC to create a specific exosome bio-signature forRCC. Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 52 is a table which lists exemplary biomarkers for cirrhosis thatcan be derived from and analyzed from exosomes specific to cirrhosis tocreate a specific exosome bio-signature for cirrhosis. Furthermore, theone or more biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 53 is a table which lists exemplary biomarkers for esophagealcancer that can be derived from and analyzed from exosomes specific toesophageal cancer to create a specific exosome bio-signature foresophageal cancer. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 54 is a table which lists exemplary biomarkers for gastric cancerthat can be derived from and analyzed from exosomes specific to gastriccancer to create a specific exosome bio-signature for gastric cancer.Furthermore, the one or more biomarkers can be present or absent,underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 55 is a table which lists exemplary biomarkers for autism that canbe derived from and analyzed from exosomes specific to autism to createa specific exosome bio-signature for autism. Furthermore, the one ormore biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 56 is a table which lists exemplary biomarkers for organ rejectionthat can be derived from and analyzed from exosomes specific to organrejection to create a specific exosome bio-signature for organrejection. Furthermore, the one or more biomarkers can be present orabsent, underexpressed or overexpressed, mutated, or modified, such asepigentically modified or post-translationally modified.

FIG. 57 is a table which lists exemplary biomarkers formethicillin-resistant staphylococcus aureus that can be derived from andanalyzed from exosomes specific to methicillin-resistant staphylococcusaureus to create a specific exosome bio-signature formethicillin-resistant staphylococcus aureus. Furthermore, the one ormore biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified, such as epigentically modified orpost-translationally modified.

FIG. 58 is a table which lists exemplary biomarkers for vulnerableplaque that can be derived from and analyzed from exosomes specific tovulnerable plaque to create a specific exosome bio-signature forvulnerable plaque. Furthermore, the one or more biomarkers can bepresent or absent, underexpressed or overexpressed, mutated, ormodified, such as epigentically modified or post-translationallymodified.

FIG. 59( a)-(i) is a table which lists exemplary gene fusions that canbe derived from, or analyzed from exosomes. The gene fusion can bebiomarker, and can be present or absent, underexpressed oroverexpressed, or modified, such as epigentically modified orpost-translationally modified.

FIG. 60( a)-(b) is a table of genes and their associated miRNAs, ofwhich the gene, such as the mRNA of the gene, their associated miRNAs,or any combination thereof, can be used as one or more biomarkers thatcan be analyzed from exosomes. Furthermore, the one or more biomarkerscan be present or absent, underexpressed or overexpressed, mutated, ormodified.

FIG. 61 is a flow chart of an exemplary method disclosed herein.

FIG. 62 illustrates a computer system that can be used in some exemplaryembodiments of the invention.

FIG. 63 illustrates results obtained from screening for proteins onexosomes, which can be used a biomarkers for the exosomes and antibodiesto the proteins can be used as binding agents. Examples of the proteinsidentified include Bcl-XL, ERCC1, Keratin 15, CD81/TAPA-1, CD9,Epithelial Specific Antigen (ESA), and Mast Cell Chymase. The one ormore biomarkers can be present or absent, underexpressed oroverexpressed, mutated, or modified.

FIG. 64 illustrates a particle based method of isolating exosomes. (A)is a schematic of a bead coated with a capture antibody, which capturesexosomes expressing that protein. In this schematic, the captureantibody is for an exosomal protein that is not specific for exosomesderived from cancer cells (“cancer exosome”). The detection antibodybinds to the captured exosome and fluoresces a signal. The detectionantibody in this example detects an antigen that is associated withcancer exosomes. (B) is an example of a screening scheme that can beperformed by multiplexing using the beads as shown in (A).

FIG. 65 depicts scanning electron micrographs (SEMs) of EpCam conjugatedbeads that have been incubated with VCaP exosomes. (A) A glass slide wascoated with poly-L-lysine and incubated with the bead solution. Afterattachment, the beads were (i) fixed sequentially with glutaraldehydeand osmium tetroxide, 30 min per fix step with a few washes in between;(ii) gradually dehydrated in acetone, 20% increments, about 5-7 min perstep; (iii) critical-point dried; and (iv) sputter-coated with gold. (B)Left: depicts a higher magnification of exosomes on an EpCam coated beadas in (A). Right: depicts exosomes isolated by ultracentrifugation andadhered to a poly-L-lysine coated glass slide and fixed and stained asin (A).

FIG. 66 is a schematic of an exosome protein expression patterns.Different proteins are typically not distributed evenly or uniformly onexosome shell. Exosome-specific proteins are typically more common,while cancer-specific proteins are less common. Exosome capture can bemore easily accomplished using a more common, less cancer-specificprotein, and cancer-specific proteins used in the detection phase.

FIG. 67 illustrates the method of depicting the results using the beadbased method of detecting exosomes from a subject. (A) For an individualpatient, a graph of the bead enumeration and signal intensity using ascreening scheme as depicted in FIG. 64B, where ˜100 capture beads areused for each capture/detection combination assay per patient. For agiven patient, the output shows number of beads detected vs. intensityof signal. The number of beads captured at a given intensity is anindication of how frequently an exosome expresses the detection proteinat that intensity. The more intense the signal for a given bead, thegreater the expression of the detection protein. (B) is a normalizedgraph obtained by combining normal patients into one curve and cancerpatients into another, and using bio-statistical analysis todifferentiate the curves. Data from each individual is normalized toaccount for variation in the number of beads read by the detectionmachine, added together, and then normalized again to account for thedifferent number of samples in each population.

FIG. 68 illustrates prostate cancer bio-signatures. (A) is a histogramof intensity values collected from a multiplexing experiment using theLuminex platform, where beads were functionalized with CD63 antibody,incubated with exosomes purified from patient plasma, and then labeledwith a phycoerythrin (PE) conjugated EpCam antibody. The darker shadedbars (blue) represent the population from 12 normal subjects and thelighter shaded bars (green) are from 7 stage 3 prostate cancer patients.(B) is a normalized graph for each of the histograms shown in (A), asdescribed in FIG. 67. The distributions are of a Gaussian fit tointensity values from the Luminex results of (A) for both prostatepatient samples and normal samples. (C) is an example of one of theprostate bio-signatures shown in (B), the CD63 versus CD63 bio-signature(upper graph) where CD63 is used as the detector and capture antibody.The lower three panels show the results of flow cytometry on threeprostate cancer cell lines (VCaP, LNcap, and 22RV1). Points above thehorizontal line indicate beads that captured exosomes with CD63 thatcontain B7H3. Beads to the right of the vertical line indicate beadsthat have captured exosomes with CD63 that have PSMA. Those beads thatare above and to the right of the lines have all three antigens. CD63 isa surface protein that is associated with exosomes, PSMA is surfaceprotein that is associated with prostate cells, and B7H3 is a surfaceprotein that is associated with aggressive cancers (specificallyprostate, ovarian, and non-small-cell lung). The combination of allthree antigens together identifies exosomes that are from cancerprostate cells. The majority of CD63 expressing prostate cancer exosomesalso have prostate-specific membrane antigen, PSMA, and B7H3 (implicatedin regulation of tumor cell migration and invasion and an indicator ofaggressive cancer as well as clinical outcome). (D) is a prostate cancerexosome topography. The upper panels show the results of capturing andlabeling with CD63, CD9, and CD81 in various combinations. Almost allpoints are in the upper right quadrant indicating that these threemarkers are highly coupled. If an exosome has one of them, it typicallyhas all three. The lower row depicts the results of capturing cell lineexosomes with B7H3 and labeling with CD63 and PSMA. Both VCaP and 22RV1show that most exosomes captured with B7H3 also have CD63, and thatthere are two populations, those with PSMA and those without. Thepresence of B7H3 may be an indication of how aggressive the cancer is,as LNcap does not have a high amount of B7H3 containing exosomes (notmany spots with CD63). LnCap is an earlier stage prostate canceranalogue cell line.

FIG. 69: illustrates colon cancer bio-signatures. (A) shows histogramsof intensity values collected from various multiplexing experimentsusing the Luminex platform, where beads were functionalized with acapture antibody, incubated with exosomes purified form patient plasma,and then labeled with a detector antibody. The darker shaded bars (blue)represent the population from normals and the lighter shaded bars(green) are from colon cancer patients. (B) shows a normalized graph foreach of the histograms shown in (A). (C) shows a histogram of intensityvalues collected from a Luminex experiment where beads wherefunctionalized with CD66 antibody (the capture antibody), incubated withexosomes purified from patient plasma, and then labeled with a PEconjugated EpCam antibody (the detector antibody). The red population isfrom 6 normals and the green is from 21 colon cancer patients. Data fromeach individual was normalized to account for variation in the number ofbeads read by the Luminex machine, added together, and then normalizedagain to account for the different number of samples in each population.

FIG. 70 illustrates multiple detectors can increase the signal ofexosome detection. (A) Median intensity values are plotted as a functionof purified exosome concentration from the VCaP cell line when labeledwith a variety of prostate specific PE conjugated antibodies. Exosomescaptured with EpCam (left graphs) or PCSA (right graphs) and the variousproteins detected by the detector antibody are listed to the right ofeach graph. In both cases the combination of CD9 and CD63 gives the bestincrease in signal over background (bottom graphs depicting percentincrease). The combination of CD9 and CD63 gave about 200% percentincrease over background. (B) further illustrates prostatecancer/prostate exosome-specific marker multiplexing improves detectionof prostate cancer cell derived exosomes. Median intensity values areplotted as a function of purified exosome concentration from the VCaPcell line when labeled with a variety of prostate specific PE conjugatedantibodies. Exosomes captured with PCSA (left) and exosomes capturedwith EpCam (right) are depicted. In both cases the combination of B7H3and PSMA gives the best increase in signal over background.

FIG. 71 illustrates a colon cancer bio-signature for colon cancer bystage, using CD63 detector and CD63 capture. The histograms ofintensities from exosomes captured with CD63 coated beads and labeledwith CD63 conjugated PE. There are 6 patients in the control group (A),4 in stage I (B), 5 in stage 11 (C), 8 in stage III (D), and 4 stage 1V(E). Data from each individual was normalized to account for variationin the number of beads read by the Luminex machine, added together, andthen normalized again to account for the different number of samples ineach population (F).

FIG. 72: illustrates colon cancer bio-signature for colon cancer bystage, using EpCam detector and CD9 capture. The histograms ofintensities are from exosomes captured with CD9 coated beads and labeledwith EpCam. There are patients in the (A) control group, (B) stage I,(C) stage II, (D) stage III, and (E) stage 1V. Data from each individualwas normalized to account for variation in the number of beads read bythe Luminex machine, added together, and then normalized again toaccount for the different number of samples in each population (F).

FIG. 73: illustrates (A) the sensitivity and specificity, and theconfidence level, for detecting prostate cancer using antibodies to thelisted proteins listed as the detector and capture antibodies. CD63,CD9, and CD81 are general exosome markers and EpCam is a cancer marker.The individual results are depicted in (B) for EpCam versus CD63, with99% confidence, 100% (n=8) cancer patient samples were different fromthe Generalized Normal Distribution and with 99% confidence, 77% (n=10)normal patient samples were not different from the Generalized NormalDistribution; (C) for CD81 versus CD63, with 99% confidence, 90% (n=5)cancer patient samples were different from the Generalized NormalDistribution; with 99% confidence, 77% (n=10) normal patient sampleswere not different from the Generalized Normal Distribution; (D) forCD63 versus CD63, with 99% confidence, 60% (n=5) cancer patient sampleswere different from the Generalized Normal Distribution; with 99%confidence, 80% (n=10) normal patient samples were not different fromthe Generalized Normal Distribution; (E) for CD9 versus CD63, with 99%confidence, 90% (n=5) cancer patient samples were different from theGeneralized Normal Distribution; with 99% confidence, 77% (n=10) normalpatient samples were not different from the Generalized NormalDistribution.

FIG. 74 illustrates (A) the sensitivity and the confidence level fordetecting colon cancer using antibodies to the listed proteins listed asthe detector and capture antibodies. CD63, CD9 are general exosomemarkers, EpCam is a cancer marker, and CD66 is a colon marker. Theindividual results are depicted in (B) for EpCam versus CD63, with 99%confidence, 95% (n=20) cancer patient samples were different from theGeneralized Normal Distribution; with 99% confidence, 100% (n=6) normalpatient samples were not different from the Generalized NormalDistribution; (C) for EpCam versus CD9, with 99% confidence, 90% (n=20)cancer patient samples were different from the Generalized NormalDistribution; with 99% confidence, 77% (n=6) normal patient samples werenot different from the Generalized Normal Distribution; (D) for CD63versus CD63, with 99% confidence, 60% (n=20) cancer patient samples weredifferent from the Generalized Normal Distribution; with 99% confidence,80% (n=6) normal patient samples were not different from the GeneralizedNormal Distribution; (E) for CD9 versus CD63, with 99% confidence, 90%(n=20) cancer patient samples were different from the Generalized NormalDistribution; with 99% confidence, 77% (n=6) normal patient samples werenot different from the Generalized Normal Distribution; (F) for CD66versus CD9, with 99% confidence, 90% (n=20) cancer patient samples weredifferent from the Generalized Normal Distribution; with 99% confidence,77% (n=6) normal patient samples were not different from the GeneralizedNormal Distribution.

FIG. 75 illustrates the capture of prostate cancer cells-derivedexosomes from plasma with EpCam by assessing TMPRSS2-ERG expression. (A)Graduated amounts of VCAP purified exosomes were spiked into normalplasma. Exosomes were isolated using Dynal beads with either EPCAMantibody or its isotype control. RNA from the exosomes was isolated andthe expression of the TMPRSS2:ERG fusion transcript was measured usingqRT-PCR. (B) VCaP purified exosomes were spiked into normal plasma andthen incubated with Dynal magnetic beads coated with either the EpCam orisotype control antibody. RNA was isolated directly from the Dynalbeads. Equal volumes of RNA from each sample were used for RT-PCR andsubsequent Taqman assays. (C) Cycle threshold (CT) differences of theSPINK1 and GAPDH transcripts between 22RV1 exosomes captured with EpCamand IgG2 isotype negative control beads. Higher CT values indicate lowertranscript expression.

FIG. 76: illustrates the top ten differentially expressed microRNAsbetween VCaP prostate cancer cell derived exosomes and normal plasmaexosomes. VCAP cell line exosomes and exosomes from normal plasma wereisolated via ultracentrifugation followed by RNA isolation. MicroRNAswere profiled using qRT-PCR analysis. Prostate cancer cell line derivedexosomes have higher levels (lower CT values) of the indicated microRNAsas depicted in the bar graph (A) and table (B).

FIG. 77 depicts a bar graph of miR-21 expression with CD9 bead capture.1 ml of plasma from prostate cancer patients, 250 ng/ml of LNCaP, ornormal purified exosomes was incubated with CD9 coated Dynal beads. TheRNA was isolated from the beads and the bead supernatant. One sample(#6) was also uncaptured for comparison. MiR-21 expression was measuredwith qRT-PCR and the mean CT values for each sample compared. CD9capture improves the detection of miR-21 in prostate cancer samples.

FIG. 78 depicts a bar graph of miR-141 expression with CD9 bead capture.The experiment was performed as in FIG. 77, with miR-141 expressionmeasured with qRT-PCR instead of miR-21.

FIG. 79 depicts a table of the sensitivity and specificity for differentprostate signatures. “Exosome” lists the threshold value or referencevalue of exosome levels, “Prostate” lists the threshold value orreference value used for prostate exosomes, “Cancer-1,” “Cancer-2,” and“Cancer-3” lists the threshold values or reference values for the threedifferent bio-signatures for prostate cancer, the “QC-1” and “QC-2”columns list the threshold values or reference values for qualitycontrol, or reliability, and the last four columns list thespecificities and sensitivities for benign prostate hyperplasia (BPH).

DETAILED DESCRIPTION

Disclosed herein are products and processes for characterizing aphenotype of an individual by analyzing exosomes. A phenotype can be anyobservable characteristic or trait of a subject, such as a disease orcondition, a disease stage or condition stage, susceptibility to adisease or condition, prognosis of a disease stage or condition, aphysiological state; or response to therapeutics. A phenotype can resultfrom a subject's gene expression as well as the influence ofenvironmental factors and the interactions between the two, as well asfrom epigenetic modifications to nucleic acid sequences

A phenotype in a subject can be characterized by obtaining a biologicalsample from said subject and analyzing one or more exosomes from thesample. For example, characterizing a phenotype for a subject orindividual may include detecting a disease or condition (includingpre-symptomatic early stage detecting), determining the prognosis,diagnosis, or theranosis of a disease or condition, or determining thestage or progression of a disease or condition. Characterizing aphenotype can also include identifying appropriate treatments ortreatment efficacy for specific diseases, conditions, disease stages andcondition stages, predictions and likelihood analysis of diseaseprogression, particularly disease recurrence, metastatic spread ordisease relapse. A phenotype can also be a clinically distinct type orsubtype of a condition or disease, such as a cancer or tumor. Phenotypedetermination can also be a determination of a physiological condition,or an assessment of organ distress or organ rejection, such aspost-transplantation. The products and processes described herein allowassessment of a subject on an individual basis, which can providebenefits of more efficient and economical decisions in treatment.

The phenotype can be a disease or condition such as listed in Table 1.For example, the phenotype can be a tumor, neoplasm, or cancer. A cancerdetected or assessed by products or processes described herein includes,but is not limited to, breast cancer, ovarian cancer, lung cancer, coloncancer, hyperplastic polyp, adenoma, colorectal cancer, high gradedysplasia, low grade dysplasia, prostatic hyperplasia, prostate cancer,melanoma, pancreatic cancer, brain cancer (such as a glioblastoma),hematological malignancy, hepatocellular carcinoma, cervical cancer,endometrial cancer, head and neck cancer, esophageal cancer,gastrointestinal stromal tumor (GIST), renal cell carcinoma (RCC) orgastric cancer. The colorectal cancer can be CRC Dukes B or Dukes C-D.The hematological malignancy can be B-Cell Chronic Lymphocytic Leukemia,B-Cell Lymphoma-DLBCL, B—Cell Lymphoma-DLBCL-germinal center-like,B-Cell Lymphoma-DLBCL-activated B-cell-like, and Burkitt's lymphoma. Thephenotype may also be a premalignant condition, such as Barrett'sEsophagus.

The phenotype can also be an inflammatory disease, immune disease, orautoimmune disease. For example, the disease may be inflammatory boweldisease (IBD), Crohn's disease (CD), ulcerative colitis (UC), pelvicinflammation, vasculitis, psoriasis, diabetes, autoimmune hepatitis,Multiple Sclerosis, Myasthenia Gravis, Type I diabetes, RheumatoidArthritis, Psoriasis, Systemic Lupus Erythematosis (SLE), Hashimoto'sThyroiditis, Grave's disease, Ankylosing Spondylitis Sjogrens Disease,CREST syndrome, Scleroderma, Rheumatic Disease, organ rejection, PrimarySclerosing Cholangitis, or sepsis.

The phenotype can also be a cardiovascular disease, such asatherosclerosis, congestive heart failure, vulnerable plaque, stroke, orischemia. The cardiovascular disease or condition can be high bloodpressure, stenosis, vessel occlusion or a thrombotic event.

The phenotype can also be a neurological disease, such as MultipleSclerosis (MS), Parkinson's Disease (PD), Alzheimer's Disease (AD),schizophrenia, bipolar disorder, depression, autism, Prion Disease,Pick's disease, dementia, Huntington disease (HD), Down's syndrome,cerebrovascular disease, Rasmussen's encephalitis, viral meningitis,neurospsychiatric systemic lupus erythematosus (NPSLE), amyotrophiclateral sclerosis, Creutzfeldt-Jacob disease,Gerstmann-Straussler-Scheinker disease, transmissible spongiformencephalopathy, ischemic reperfusion damage (e.g. stroke), brain trauma,microbial infection, or chronic fatigue syndrome. The phenotype may alsobe a condition such as fibromyalgia, chronic neuropathic pain, orperipheral neuropathic pain.

The phenotype may also be an infectious disease, such as a bacterial,viral or yeast infection. For example, the disease or condition may beWhipple's Disease, Prion Disease, cirrhosis, methicillin-resistantstaphylococcus aureus, HIV, hepatitis, syphilis, meningitis, malaria,tuberculosis, or influenza. Viral proteins, such as HIV or HCV-likeparticles can be assessed in an exosome, to characterize a viralcondition.

The phenotype can also be a perinatal or pregnancy related condition(e.g. preeclampsia or preterm birth), metabolic disease or condition,such as a metabolic disease or condition associated with ironmetabolism. For example, hepcidin can be assayed in an exosome tocharacterize an iron deficiency. The metabolic disease or condition canalso be diabetes, inflammation, or a perinatal condition.

Subject

One or more phenotypes of a subject can be determined by analyzingexosomes in a biological sample obtained from the subject. A subject orpatient can include, but is not limited to, mammals such as bovine,avian, canine, equine, feline, ovine, porcine, or primate animals(including humans and non-human primates). A subject may also includemammals of importance due to being endangered, such as Siberian tigers;or economic importance, such as animals raised on farms for consumptionby humans, or animals of social importance to humans such as animalskept as pets or in zoos. Examples of such animals include but are notlimited to: carnivores such as cats and dogs; swine including pigs, hogsand wild boars; ruminants or ungulates such as cattle, oxen, sheep,giraffes, deer, goats, bison, camels or horses. Also included are birdsthat are endangered or kept in zoos, as well as fowl and moreparticularly domesticated fowl, i.e. poultry, such as turkeys andchickens, ducks, geese, guinea fowl. Also included are domesticatedswine and horses (including race horses). In addition, any animalspecies connected to commercial activities are also included such asthose animals connected to agriculture and aquaculture and otheractivities in which disease monitoring, diagnosis, and therapy selectionare routine practice in husbandry for economic productivity and/orsafety of the food chain.

The subject can have a pre-existing disease or condition, such ascancer. Alternatively, the subject may not have any known pre-existingcondition. The subject may also be non-responsive to an existing or pasttreatment, such as a treatment for cancer.

Samples

The biological sample obtained from the subject may be any bodily fluid.For example, the biological sample can be peripheral blood, sera,plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bonemarrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breastmilk, broncheoalveolar lavage fluid, semen (including prostatic fluid),Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecalmatter, hair, tears, cyst fluid, pleural and peritoneal fluid,pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid,menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stoolwater, pancreatic juice, lavage fluids from sinus cavities,bronchopulmonary aspirates or other lavage fluids. A biological samplemay also include the blastocyl cavity, umbilical cord blood, or maternalcirculation which may be of fetal or maternal origin. The biologicalsample may also be a tissue sample or biopsy, from which exosomes may beobtained. For example, if the sample is a solid sample, cells from thesample can be cultured and exosome product induced (see for example,Example 1).

Table 1 provides a list of examples of diseases, conditions, orbiological states and a corresponding list of biological samples fromwhich exosomes may be analyzed.

TABLE 1 Examples of Biological Samples for Exosome Analysis for VariousDiseases, Conditions, or Biological States Disease, Condition orBiological State Biological Samples Cancers/neoplasms affecting thefollowing tissue Blood, serum, cerebrospinal fluid (CSF), urine, sputum,types/bodily systems: breast, lung, ovarian, colon, rectal, ascites,synovial fluid, semen, nipple aspirates, saliva, prostate, pancreatic,brain, bone, connective tissue, glands, bronchoalveolar lavage fluid,tears, oropharyngeal washes, skin, lymph, nervous system, endocrine,germ cell, feces, peritoneal fluids, pleural effusion, sweat, tears,genitourinary, hematologic/blood, bone marrow, muscle, aqueous humor,pericardial fluid, lymph, chyme, chyle, eye, esophageal, fat tissue,thyroid, pituitary, spinal cord, bile, stool water, amniotic fluid,breast milk, pancreatic bile duct, heart, gall bladder, bladder, testes,cervical, juice, cerumen, Cowper's fluid or pre-ejaculatory fluid,endometrial, renal, ovarian, digestive/gastrointestinal, femaleejaculate, interstitial fluid, menses, mucus, pus, stomach, head andneck, liver, leukemia, sebum, vaginal lubrication, vomitrespiratory/thorasic, cancers of unknown primaryNeurodegenerative/neurological disorders: Parkinson's Blood, serum, CSF,urine disease, Alzheimer's Disease and multiple sclerosis,Schizophrenia, and bipolar disorder, spasticity disorders, epilepsyCardiovascular Disease: atherosclerosis, cardiomyopathy, Blood, serum,CSF, urine endocarditis, vunerable plaques, infection Stroke: ischemic,intracerebral hemorrhage, subarachnoid Blood, serum, CSF, urinehemorrhage, transient ischemic attacks (TIA) Pain disorders: peripheralneuropathic pain and chronic Blood, serum, CSF, urine neuropathic pain,and fibromyalgia, Autoimmune disease: systemic and localized diseases,Blood, serum, CSF, urine, synovial fluid rheumatic disease, Lupus,Sjogren's syndrome Digestive system abnormalities: Barrett's esophagus,Blood, serum, CSF, urine irritable bowel syndrome, ulcerative colitis,Crohn's disease, Diverticulosis and Diverticulitis, Celiac DiseaseEndocrine disorders: diabetes mellitus, various forms of Blood, serum,CSF, urine Thyroiditis,, adrenal disorders, pituitary disorders Diseasesand disorders of the skin: psoriasis Blood, serum, CSF, urine, synovialfluid, tears Urological disorders: benign prostatic hypertrophy (BPH),Blood, serum, urine polycystic kidney disease, interstitial cystitisHepatic disease/injury: Cirrhosis, induced hepatotoxicity Blood, serum,urine (due to exposure to natural or synthetic chemical sources) Kidneydisease/injury: acute, sub-acute, chronic Blood, serum, urineconditions, Podocyte injury, focal segmental glomerulosclerosisEndometriosis Blood, serum, urine, vaginal fluids Osteoporosis Blood,serum, urine, synovial fluid Pancreatitis Blood, serum, urine,pancreatic juice Asthma Blood, serum, urine, sputum, bronchiolar lavagefluid Allergies Blood, serum, urine, sputum, bronchiolar lavage fluidPrion-related diseases Blood, serum, CSF, urine Viral Infections:HIV/AIDS Blood, serum, urine Sepsis Blood, serum, urine, tears, nasallavage Organ rejection/transplantation Blood, serum, urine, variouslavage fluids Differentiating conditions: adenoma versus hyperplasticBlood, serum, urine, sputum, feces, colonic lavage fluid polyp,irritable bowel syndrome (IBS) versus normal, classifying Dukes stagesA, B, C, and/or D of colon cancer, adenoma with low-grade hyperplasiaversus high- grade hyperplasia, adenoma versus normal, colorectal cancerversus normal, IBS versus. ulcerative colitis (UC) versus Crohn'sdisease (CD), Pregnancy related physiological states, conditions, orMaternal serum, amniotic fluid, cord blood affiliated diseases: geneticrisk, adverse pregnancy outcomes

The biological samples may be obtained through a third party, such as aparty not performing the analysis of the exosome. For example, thesample may be obtained through a clinician, physician, or other healthcare manager of a subject from which the sample is derived.Alternatively, the biological sample may obtained by the same partyanalyzing the exosomes.

The volume of the biological sample used for analyzing an exosome can bein the range of between 0.1-20 mL, such as less than about 20, 15, 10,9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.1 mL.

Furthermore, analysis of one or more exosomes in a biological sample canbe used to determine whether an additional biological sample should beobtained for analysis. For example, analysis of one or more exosomes ina serum sample can be used to determine whether a biopsy should beobtained.

Exosomes

The exosomes from a biological sample for analysis are used to determinea phenotype. Exosomes are small vesicles that are released into theextracellular environment from a variety of different cells such as butnot limited to, cells that originate from, or are derived from, theectoderm, endoderm, or mesoderm including any such cells that haveundergone genetic, environmental, and/or any other variations oralterations (e.g. Tumor cells or cells with genetic mutations). Anexosome is typically created intracellularly when a segment of the cellmembrane spontaneously invaginates and is ultimately exocytosed (see forexample, Keller et al., Immunol. Lett. 107 (2): 102-8 (2006)). Exosomescan have, but not be limited to, a diameter of greater than about 10,20, or 30 nm. They can have a diameter of about 30-1000 nm, about 30-800nm, about 30-200 nm, or about 30-100 nm. In some embodiments, theexosomes can have, but not be limited to, a diameter of less than about10,000 nm, 1000 nm, 800 nm, 500 nm, 200 nm, 100 nm or 50 nm. As usedthroughout, the term “about,” when referring to a value or to an amountis meant to encompass variations in some embodiments±10% from thespecified amount, as such variations are appropriate.

Exosomes may also be referred to as microvesicles, nanovesicles,vesicles, dexosomes, bleb, blebby, prostasomes, microparticles,intralumenal vesicles, endosomal-like vesicles or exocytosed vehicles.As used herein, exosomes can also include any shed membrane boundparticle that is derived from either the plasma membrane or an internalmembrane. Exosomes can also include cell-derived structures bounded by alipid bilayer membrane arising from both herniated evagination(blebbing) separation and sealing of portions of the plasma membrane orfrom the export of any intracellular membrane-bounded vesicularstructure containing various membrane-associated proteins of tumororigin, including surface-bound molecules derived from the hostcirculation that bind selectively to the tumor-derived proteins togetherwith molecules contained in the exosome lumen, including but not limitedto tumor-derived microRNAs or intracellular proteins. Blebs and blebbingare further described in Charras et al., Nature Reviews Molecular andCell Biology, Vol. 9, No. 11, p. 730-736 (2008). Exosomes can alsoinclude membrane fragments. Circulating tumor-derived exosomes (CTEs) asreferenced herein are exosomes that are shed into circulation or bodilyfluids from tumor cells. CTEs, as with cell-of-origin specific exosomes,typically have unique biomarkers that permit their isolation from bodilyfluids in a highly specific manner.

Exosomes can be directly assayed from the biological samples, such thatthe level of exosomes is determined or the one or more biomarkers of theexosomes is determined without prior isolation, purification, orconcentration of the exosomes. Alternatively, exosomes may be isolated,purified, or concentrated from a sample prior to analysis.

Isolation of Exosomes

An exosome may be purified or concentrated prior to analysis. Analysisof an exosome can include quantitiating the amount one or more exosomepopulations of a biological sample. For example, a heterogeneouspopulation of exosomes can be quantitated, or a homogeneous populationof exosomes, such as a population of exosomes with a particularbiomarker profile, a particular bio-signature, or derived from aparticular cell type (cell-of-origin specific exosomes) can be isolatedfrom a heterogeneous population of exosomes and quantitated. Analysis ofan exosome can also include detecting, quantitatively or qualitatively,a particular biomarker profile or a bio-signature, of an exosome, asdescribed below.

An exosome can be stored and archived, such as in a bio-fluid bank andretrieved for analysis as necessary. An exosome may also be isolatedfrom a biological sample that has been previously harvested and storedfrom a living or deceased subject. In addition, an exosome may beisolated from a biological sample which has been collected as describedin King et al., Breast Cancer Res 7(5): 198-204 (2005). An exosome maybe isolated from an archived or stored sample. Alternatively, an exosomemay be isolated from a biological sample and analyzed without storing orarchiving of the sample. Furthermore, a third party may obtain or storethe biological sample, or obtain or store the exosomes for analysis.

An enriched population of exosomes can be obtained from a biologicalsample. For example, exosomes may be concentrated or isolated from abiological sample using size exclusion chromatography, density gradientcentrifugation, differential centrifugation, nanomembraneultrafiltration, immunoabsorbent capture, affinity purification,microfluidic separation, or combinations thereof.

Size exclusion chromatography, such as gel permeation columns,centrifugation or density gradient centrifugation, and filtrationmethods can be used. For example, exosomes can be isolated bydifferential centrifugation, anion exchange and/or gel permeationchromatography (for example, as described in U.S. Pat. Nos. 6,899,863and 6,812,023), sucrose density gradients, organelle electrophoresis(for example, as described in U.S. Pat. No. 7,198,923), magneticactivated cell sorting (MACS), or with a nanomembrane ultrafiltrationconcentrator. Various combinations of isolation or concentration methodscan be used.

Highly abundant proteins, such as albumin and immunoglobulin, may hinderisolation of exosomes from a biological sample. For example, exosomesmay be isolated from a biological sample using a system that utilizesmultiple antibodies that are specific to the most abundant proteinsfound in blood. Such a system can remove up to several proteins at once,thus unveiling the lower abundance species such as cell-of-originspecific exosomes.

This type of system can be used for isolation of exosomes frombiological samples such as blood, cerebrospinal fluid or urine. Theisolation of exosomes from a biological sample may also be enhanced byhigh abundant protein removal methods as described in Chromy et al. J.Proteome Res 2004; 3:1120-1127. In another embodiment, the isolation ofexosomes from a biological sample may also be enhanced by removing serumproteins using glycopeptide capture as described in Zhang et al, MolCell Proteomics 2005; 4:144-155. In addition, exosomes from a biologicalsample such as urine may be isolated by differential centrifugationfollowed by contact with antibodies directed to cytoplasmic oranti-cytoplasmic epitopes as described in Pisitkun et al., Proc NatlAcad Sci USA, 2004; 101:13368-13373.

Isolation or enrichment of exosomes from biological samples can also beenhanced by use of sonication (for example, by applying ultrasound), orthe use of detergents, other membrane-active agents, or any combinationthereof. For example, ultrasonic energy can be applied to a potentialtumor site, and without being bound by theory, release of exosomes fromthe tissue can be increased, allowing an enriched population of exosomesthat can be analyzed or assessed from a biological sample using one ormore methods disclosed herein.

Binding Agents

A binding agent is an agent that binds to an exosomal component, such asa biomarker of an exosome. The binding agent can be a capture agent. Acapture agent captures the exosome by binding to an exosomal target,such as a biomarker on the exosome. For example, the capture agent canbe a capture antibody that binds to an antigen on the exosome. Thecapture agent can be coupled to a substrate and used to isolate theexosome, such as described herein.

A binding agent can be used after exosomes are concentrated or isolatedfrom a biological sample. For example, exosomes can first be isolatedfrom a biological sample before exosomes with a specific biomarker areisolated using a binding agent for the biomarker. Thus, exosomes withthe specific biomarker is isolated from a heterogeneous population ofexosomes. Alternatively, a binding agent may be used on a biologicalsample comprising exosomes without a prior isolation step orconcentration of exosomes. For example, a binding agent is used toisolate an exosome with a specific biomarker from a biological sample.

A binding agent can be DNA, RNA, monoclonal antibodies, polyclonalantibodies, Fabs, Fab′, single chain antibodies, synthetic antibodies,aptamers (DNA/RNA), peptoids, zDNA, peptide nucleic acids (PNAs), lockednucleic acids (LNAs), lectins, synthetic or naturally occurring chemicalcompounds (including but not limited to drugs, labeling reagents),dendrimers, or combinations thereof. For example, the binding agent canbe a capture antibody.

In some instances, a single binding agent can be employed to isolate anexosome. In other instances, a combination of different binding agentsmay be employed to isolate an exosome. For example, at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75 or100 different binding agents may be used to isolate an exosome from abiological sample. Furthermore, the one or more different binding agentsfor an exosome can form the bio-signature of the exosome, furtherdescribed below.

Different binding agents can also be used for multiplexing. For example,isolation of more than one population of exosomes (for example, exosomesfrom specific cell types) can be performed by isolating each exosomepopulation with a different binding agent. Different binding agents canbe bound to different particles, wherein the different particles arelabeled. In another embodiment, an array comprising different bindingagents can be used for multiplex analysis, wherein the different bindingagents are differentially labeled or can be ascertained based on thelocation of the binding agent on the array. Multiplexing can beaccomplished up to the resolution capability of the labels or detectionmethod, such as described below.

The binding agent can be an antibody. For example, an exosome may beisolated using one or more antibodies specific for one or more antigenspresent on the exosome. For example, an exosome can have CD63 on itssurface, and an antibody, or capture antibody, for CD63 can be used toisolate the exosome. Alternatively, an exosome derived from a tumor cellcan express EpCam, the exosome can be isolated using an antibody forEpCam and CD63. Other antibodies for isolating exosomes can include anantibody, or capture antibody, to CD9, PSCA, TNFR, CD63, B7H3, MFG-E8,EpCam, Rab, CD81, STEAP, PCSA, PSMA, or 5T4.

The antibodies disclosed herein can be immunoglobulin molecules orimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsan antigen and synthetic antibodies. The immunoglobulin molecules can beof any class (e.g., IgG, IgE, IgM, IgD or IgA) or subclass ofimmunoglobulin molecule. Antibodies include, but are not limited to,polyclonal, monoclonal, bispecific, synthetic, humanized and chimericantibodies, single chain antibodies, Fab fragments and F(ab′)₂fragments, Fv or Fv′ portions, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, or epitope-bindingfragments of any of the above. An antibody, or generally any molecule,“binds specifically” to an antigen (or other molecule) if the antibodybinds preferentially to the antigen, and, e.g., has less than about 30%,20%, 10%, 5% or 1% cross-reactivity with another molecule.

The binding agent can also be a polypeptide or peptide. Polypeptide isused in its broadest sense and may include a sequence of subunit aminoacids, amino acid analogs, or peptidomimetics. The subunits may belinked by peptide bonds. The polypeptides may be naturally occurring,processed forms of naturally occurring polypeptides (such as byenzymatic digestion), chemically synthesized or recombinantly expressed.The polypeptides for use in the methods of the present invention may bechemically synthesized using standard techniques. The polypeptides maycomprise D-amino acids (which are resistant to L-amino acid-specificproteases), a combination of D- and L-amino acids, β amino acids, orvarious other designer or non-naturally occurring amino acids (e.g.,(β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids,etc.) to convey special properties. Synthetic amino acids may includeornithine for lysine, and norleucine for leucine or isoleucine. Inaddition, the polypeptides can have peptidomimetic bonds, such as esterbonds, to prepare polypeptides with novel properties. For example, apolypeptide may be generated that incorporates a reduced peptide bond,i.e., R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues orsequences. A reduced peptide bond may be introduced as a dipeptidesubunit. Such a polypeptide would be resistant to protease activity, andwould possess an extended half-live in vivo. Polypeptides can alsoinclude peptoids (N-substituted glycines), in which the side chains areappended to nitrogen atoms along the molecule's backbone, rather than tothe α-carbons, as in amino acids. Polypeptides and peptides are intendedto be used interchangeably throughout this application, i.e. where theterm peptide is used, it may also include polypeptides and where theterm polypeptides is used, it may also include peptides.

An exosome may be isolated using a known binding agent. For example, thebinding agent can be an agent that binds exosomal “housekeepingproteins,” or general exosome biomarkers, such as CD63, CD9, CD81, CD82,CD37, CD53, or Rab-5b. The binding agent can also be an agent that bindsto exosomes derived from specific cell types, such as tumor cells (e.g.binding agent for EpCam) or specific cell-of-origins, such as describedbelow. For example, the binding agent used to isolate an exosome may bea binding agent for an antigen selected from FIG. 1. The binding agentfor an exosome can also be selected from those listed in FIG. 2. Forexample, the binding agent can be for an antigen such as 5T4, B7H3,caveolin, CD63, CD9, E-Cadherin, MFG-E8, PSCA, PSMA, Rab-5B, STEAP,TNFR1, CD81, EpCam, CD59, or CD66. One or more binding agents, such asone or more binding agents for two or more of the antigens, can be usedfor isolating an exosome. The binding agent used can be selected basedon the desire of isolating exosomes derived from particular cell types,or cell-of-origin specific exosomes.

A binding agent can also be linked directly or indirectly to a solidsurface or substrate. A solid surface or substrate can be any physicallyseparable solid to which a binding agent can be directly or indirectlyattached including, but not limited to, surfaces provided by microarraysand wells, particles such as beads, columns, optical fibers, wipes,glass and modified or functionalized glass, quartz, mica, diazotizedmembranes (paper or nylon), polyformaldehyde, cellulose, celluloseacetate, paper, ceramics, metals, metalloids, semiconductive materials,quantum dots, coated beads or particles, other chromatographicmaterials, magnetic particles; plastics (including acrylics,polystyrene, copolymers of styrene or other materials, polypropylene,polyethylene, polybutylene, polyurethanes, TEFLON™, etc.),polysaccharides, nylon or nitrocellulose, resins, silica or silica-basedmaterials including silicon and modified silicon, carbon, metals,inorganic glasses, plastics, ceramics, conducting polymers (includingpolymers such as polypyrole and polyindole); micro or nanostructuredsurfaces such as nucleic acid tiling arrays, nanotube, nanowire, ornanoparticulate decorated surfaces; or porous surfaces or gels such asmethacrylates, acrylamides, sugar polymers, cellulose, silicates, orother fibrous or stranded polymers. In addition, as is known the art,the substrate may be coated using passive or chemically-derivatizedcoatings with any number of materials, including polymers, such asdextrans, acrylamides, gelatins or agarose. Such coatings can facilitatethe use of the array with a biological sample.

For example, an antibody used to isolate an exosome can be bound to asolid substrate such as a well, such as commercially available plates(e.g. from Nunc, Milan Italy). Each well can be coated with theantibody. In some embodiments, the antibody used to isolate an exosomecan be bound to a solid substrate such as an array. The array can have apredetermined spatial arrangement of molecule interactions, bindingislands, biomolecules, zones, domains or spatial arrangements of bindingislands or binding agents deposited within discrete boundaries. Further,the term array may be used herein to refer to multiple arrays arrangedon a surface, such as would be the case where a surface bore multiplecopies of an array. Such surfaces bearing multiple arrays may also bereferred to as multiple arrays or repeating arrays.

A binding agent can also be bound to particles such as beads ormicrospheres. For example, an antibody specific for an exosomalcomponent can be bound to a particle, and the antibody-bound particle isused to isolate exosomes from a biological sample. In some embodiments,the microspheres may be magnetic or fluorescently labeled. In addition,a binding agent for isolating exosomes can be a solid substrate itself.For example, latex beads, such as aldehyde/sulfate beads (InterfacialDynamics, Portland, Oreg.) can be used.

A binding agent bound to a magnetic bead can also be used to isolate anexosome. For example, a biological sample such as serum from a patientcan be collected for colon cancer screening. The sample can be incubatedwith anti-CCSA-3 (Colon Cancer-Specific Antigen) coupled to magneticmicrobeads. A low-density microcolumn can be placed in the magneticfield of a MACS Separator and the column is then washed with a buffersolution such as Tris-buffered saline. The magnetic immune complexes canthen be applied to the column and unbound, non-specific material can bediscarded. The CCSA-3 selected exosomes can be recovered by removing thecolumn from the separator and placing it on a collection tube. A buffercan be added to the column and the magnetically labeled exosomes can bereleased by applying the plunger supplied with the column. The isolatedexosomes can be diluted in IgG elution buffer and the complex can thenbe centrifuged to separate the microbeads from the exosomes. Thepelleted isolated cell-of-origin specific exosomes can be resuspended inbuffer such as phosphate-buffered saline and quantitated. Alternatively,due to the strong adhesion force between the antibody capturedcell-of-origin specific exosomes and the magnetic microbeads, aproteolytic enzyme such as trypsin can be used for the release ofcaptured exosomes without the need for centrifugation. The proteolyticenzyme can be incubated with the antibody captured cell-of-originspecific exosomes for at least a time sufficient to release theexosomes.

A binding agent, such as an antibody, for isolating an exosome ispreferably contacted with the biological sample comprising the exosomeof interest for at least a time sufficient for the binding agent to bindto an exosomal component. For example, an antibody may be contacted witha biological sample for various intervals ranging from seconds days,including but not limited to, about 10 minutes, 30 minutes, 1 hour, 3hours, 5 hours, 7 hours, 10 hours, 15 hours, 1 day, 3 days, 7 days or 10days.

A binding agent, such as an antibody specific to an antigen listed inFIG. 1, or a binding agent listed in FIG. 2, can be labeled with,including but not limited to, a magnetic label, a fluorescent moiety, anenzyme, a chemiluminescent probe, a metal particle, a non-metalcolloidal particle, a polymeric dye particle, a pigment molecule, apigment particle, an electrochemically active species, semiconductornanocrystal or other nanoparticles including quantum dots or goldparticles. The label can be, but not be limited to, fluorophores,quantum dots, or radioactive labels. For example, the label can be aradioisotope (radionuclides), such as ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu,⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, or²¹³Bi. The label can be a fluorescent label, such as a rare earthchelate (europium chelate), fluorescein type, such as, but not limitedto, FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; a rhodamine type,such as, but not limited to, TAMRA; dansyl; Lissamine; cyanines;phycoerythrins; Texas Red; and analogs thereof.

A binding agent can be directly or indirectly labeled, e.g., the labelis attached to the antibody through biotin-streptavidin. Alternatively,an antibody is not labeled, but is later contacted with a secondantibody that is labeled after the first antibody is bound to an antigenof interest.

For example, various enzyme-substrate labels are available or disclosed(see for example, U.S. Pat. No. 4,275,149). The enzyme generallycatalyzes a chemical alteration of a chromogenic substrate that can bemeasured using various techniques. For example, the enzyme may catalyzea color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Examples ofenzymatic labels include luciferases (e.g., firefly luciferase andbacterial luciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Examples ofenzyme-substrate combinations include, but are not limited to,horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethylbenzidinehydrochloride (TMB)); alkaline phosphatase (AP) with para-nitrophenylphosphate as chromogenic substrate; and β-D-galactosidase ((β-D-Gal)with a chromogenic substrate (e.g., p-nitrophenyl-(β-D-galactosidase) orfluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Depending on the method of isolation used, the binding agent may belinked to a solid surface or substrate, such as arrays, particles, wellsand other substrates described above. Methods for direct chemicalcoupling of antibodies, to the cell surface are known in the art, andmay include, for example, coupling using glutaraldehyde or maleimideactivated antibodies. Methods for chemical coupling using multiple stepprocedures include biotinylation, coupling of trinitrophenol (TNP) ordigoxigenin using for example succinimide esters of these compounds.Biotinylation can be accomplished by, for example, the use ofD-biotinyl-N-hydroxysuccinimide. Succinimide groups react effectivelywith amino groups at pH values above 7, and preferentially between aboutpH 8.0 and about pH 8.5. Biotinylation can be accomplished by, forexample, treating the cells with dithiothreitol followed by the additionof biotin maleimide.

Flow Cytometry

Isolation of exosomes using particles such as beads or microspheres canalso be performed using flow cytometry. Flow cytometry can be used forsorting microscopic particles suspended in a stream of fluid. Asparticles pass through they can be selectively charged and on their exitcan be deflected into separate paths of flow. It is therefore possibleto separate populations from an original mix, such as a biologicalsample, with a high degree of accuracy and speed. Flow cytometry allowssimultaneous multiparametric analysis of the physical and/or chemicalcharacteristics of single cells flowing through an optical/electronicdetection apparatus. A beam of light, usually laser light, of a singlefrequency (color) is directed onto a hydrodynamically focused stream offluid. A number of detectors are aimed at the point where the streampasses through the light beam; one in line with the light beam (ForwardScatter or FSC) and several perpendicular to it (Side Scatter or SSC)and one or more fluorescent detectors.

Each suspended particle passing through the beam scatters the light insome way, and fluorescent chemicals in the particle may be excited intoemitting light at a lower frequency than the light source. Thiscombination of scattered and fluorescent light is picked up by thedetectors, and by analyzing fluctuations in brightness at each detector(one for each fluorescent emission peak), it is possible to deducevarious facts about the physical and chemical structure of eachindividual particle. FSC correlates with the cell size and SSC dependson the inner complexity of the particle, such as shape of the nucleus,the amount and type of cytoplasmic granules or the membrane roughness.Some flow cytometers have eliminated the need for fluorescence and useonly light scatter for measurement.

Flow cytometers can analyze several thousand particles every second in“real time” and can actively separate out and isolate particles havingspecified properties. They offer high-throughput automatedquantification, and separation, of the set parameters for a high numberof single cells during each analysis session. Modern instruments havemultiple lasers and fluorescence detectors, for example up to 4 lasersand 18 fluorescence detectors, allowing multiple labels to be used tomore precisely specify a target population by their phenotype.

The data resulting from flow-cytometers can be plotted in 1 dimension toproduce histograms or seen in 2 dimensions as dot plots or in 3dimensions with newer software. The regions on these plots can besequentially separated by a series of subset extractions which aretermed gates. Specific gating protocols exist for diagnostic andclinical purposes especially in relation to hematology. The plots areoften made on logarithmic scales. Because different fluorescent dye'semission spectra overlap, signals at the detectors have to becompensated electronically as well as computationally. Fluorophores forlabeling biomarkers may include those described in Ormerod, FlowCytometry 2nd ed., Springer-Verlag, New York (1999), and in Nida et al.,Gynecologic Oncology 2005; 4 889-894 which is incorporated herein byreference.

Multiplexing

Different binding agents can be used for multiplexing different exosomepopulations. Different exosome populations can be isolated or detectedusing different binding agents. Each exosome population in a biologicalsample can be labeled with a different signaling label, such asfluorophores, quantum dots, or radioactive labels, such as describedabove. The label can be directly conjugated to a binding agent orindirectly used to detect a binding agent. The number of populationsdetected in a multiplexing assay is dependent on the resolutioncapability of the labels and the summation of signals, as more than twodifferentially labeled exosome populations that bind two or moreaffinity elements can produce summed signals.

Multiplexing of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 50, 75 or 100 different exosome populations maybe performed. For example, one population of exosomes specific to acell-of-origin can be assayed along with a second population of exosomesspecific to a different cell-of-origin, where each population is labeledwith a different label. Alternatively, a population of exosomes with aparticular biomarker or bio-signature can be assayed along with a secondpopulation of exosomes with a different biomarker or bio-signature.

In one embodiment, multiplex analysis is performed by applying aplurality of exosomes comprising more than one population of exosomes toa plurality of substrates, such as beads. Each bead is coupled to one ormore capture agents. The plurality of beads is divided into subsets,where beads with the same capture agent or combination of capture agentsform a subset of beads, such that each subset of beads has a differentcapture agent or combination of capture agents than another subset ofbeads. The beads can then be used to capture exosomes that comprises acomponent that binds to the capture agent. The different subsets can beused to capture different populations of exosomes. The captured exosomescan then be analyzed by detecting one or more biomarkers of theexosomes.

Flow cytometry can be used in combination with a particle-based or beadbased assay. Multiparametric immunoassays or other high throughputdetection assays using bead coatings with cognate ligands and reportermolecules with specific activities consistent with high sensitivityautomation can be used. For example, beads in each subset can bedifferentially labeled from another subset. For example, in a particlebased assay system, a binding agent or capture agent for an exosome,such as a capture antibody, can be immobilized on addressable beads ormicrospheres. Each binding agent for each individual binding assay (suchas an immunoassay when the binding agent is an antibody) can be coupledto a distinct type of microsphere (i.e., microbead) and the bindingassay reaction takes place on the surface of the microspheres.Microspheres can be distinguished by different labels, for example, amicrosphere with a specific capture agent would have a differentsignaling label as compared to another microsphere with a differentcapture agent. For example, microspheres can be dyed with discretefluorescence intensities such that the fluorescence intensity of amicrosphere with a specific binding agent is different than that ofanother microsphere with a different binding agent.

The microsphere can be labeled or dyed with at least 2 different labelsor dyes. In some embodiments, the microsphere is labeled with at least3, 4, 5, 6, 7, 8, 9, or 10 different labels. Different microspheres in aplurality of microspheres can have more than one label or dye, whereinvarious subsets of the microspheres have various ratios and combinationsof the labels or dyes permitting detection of different microsphereswith different binding agents. For example, the various ratios andcombinations of labels and dyes can permit different fluorescentintensities. Alternatively, the various ratios and combinations maybeused to generate different detection patters to identify the bindingagent. The microspheres can be labeled or dyed externally or may haveintrinsic fluorescence or signaling labels. Beads can be loadedseparately with their appropriate binding agents and thus, differentexosome populations can be isolated based on the different bindingagents on the differentially labeled microspheres to which the differentbinding agents are coupled.

In another embodiment, multiplex analysis can be performed using aplanar substrate, wherein the said substrate comprises a plurality ofcapture agents. The plurality of capture agents can capture one or morepopulations of exosomes, and one or more biomarkers of the capturedexosomes detected. The planar substrate can be a microarray or othersubstrate as further described herein.

Novel Binding Agents

An exosome may be isolated using a binding agent for a novel componentof an exosome, such as an antibody for a novel antigen specific to anexosome of interest. Novel antigens that are specific to exosomes ofinterest may be isolated or identified using different test compounds ofknown composition bound to a substrate, such as an array or a pluralityof particles, which can allow a large amount of chemical/structuralspace to be adequately sampled using only a small fraction of the space.The novel antigen identified can also serve as a biomarker for theexosome. For example, a novel antigen identified for a cell-of-originspecific exosome can be a biomarker for that particular cell-of-originspecific exosome.

A binding agent can be identified by screening either a homogeneous orheterogeneous exosome population against test compounds. Since thecomposition of each test compound on the substrate surface is known,this constitutes a screen for affinity elements. For example, a testcompound array comprises test compounds at specific locations on thesubstrate addressable locations, and can be used to identify one or morebinding agents for an exosome. The test compounds can all be unrelatedor related based on minor variations of a core sequence or structure.The different test compounds may include variants of a given testcompound (such as polypeptide isoforms), test compounds that arestructurally or compositionally unrelated, or a combination thereof.

Test compounds can be peptoids, polysaccharides, organic compounds,inorganic compounds, polymers, lipids, nucleic acids, polypeptides,antibodies, proteins, polysaccharides, or other compounds. The testcompounds can be natural or synthetic. The test compounds can compriseor consist of linear or branched heteropolymeric compounds based on anyof a number of linkages or combinations of linkages (e.g., amide, ester,ether, thiol, radical additions, metal coordination, etc.), dendriticstructures, circular structures, cavity structures or other structureswith multiple nearby sites of attachment that serve as scaffolds uponwhich specific additions are made. These test compounds can be spottedon the substrate or synthesized in situ, using standard methods in theart. In addition, the test compounds can be spotted or synthesized insitu in combinations in order to detect useful interactions, such ascooperative binding.

The test compounds can be polypeptides with known amino acid sequences,thus, detection a test compound binding with an exosome can lead toidentification of a polypeptide of known amino sequence that can be usedas a binding agent. For example, a homogenous population of exosomes canbe applied to a spotted array on a slide containing between a few and1,000,000 test polypeptides having a length of variable amino acids. Thepolypeptides can be attached to the surface through the C-terminus. Thesequence of the polypeptides can be generated randomly from 19 aminoacids, excluding cysteine. The binding reaction can include anon-specific competitor, such as excess bacterial proteins labeled withanother dye such that the specificity ratio for each polypeptide bindingtarget can be determined. The polypeptides with the highest specificityand binding can be selected. The identity of the polypeptide on eachspot is known, and thus can be readily identified. Once the novelantigens specific to the homogeneous exosome population, such as acell-of-origin specific exosome is identified, such cell-of-originspecific exosomes may subsequently be isolated using such antigens inmethods described hereafter.

Arrays can also be used for identifying antibodies for isolatingexosomes. Test antibodies can be attached to an array and screenedagainst a heterogeneous population of exosomes to identify antibodiesthat can be used to isolate, and identify, an exosome. A homogeneouspopulation of exosomes, such as cell-of-origin specific exosomes, canalso be screened with an antibody array. Other than identifyingantibodies to isolate the homogeneous population of exosomes, one ormore protein biomarkers specific to the homogenous exosome populationcan be identified. Commercially available platforms with test antibodiespre-selected, or custom selection of test antibodies attached to thearray, can be used. For example, an antibody array from Full MoonBiosystems can be screened using prostate cancer cell derived exosomes,identifying antibodies to Bcl-XL, ERCC1, Keratin 15, CD81/TAPA-1, CD9,Epithelial Specific Antigen (ESA), and Mast Cell Chymase as bindingagents (see for example, FIG. 63), and the proteins identified can beused as biomarkers for the exosomes.

An antibody or synthetic antibody to be used as a binding agent can alsobe identified through a peptide array. Another method is the use ofsynthetic antibody generation through antibody phage display. M13bacteriophage libraries of antibodies (e.g. Fabs) are displayed on thesurfaces of phage particles as fusions to a coat protein. Each phageparticle displays a unique antibody and also encapsulates a vector thatcontains the encoding DNA. Highly diverse libraries can be constructedand represented as phage pools, which can be used in antibody selectionfor binding to immobilized antigens. Antigen-binding phages are retainedby the immobilized antigen, and the nonbinding phages are removed bywashing. The retained phage pool can be amplified by infection of anEscherichia coli host and the amplified pool can be used for additionalrounds of selection to eventually obtain a population that is dominatedby antigen-binding clones. At this stage, individual phase clones can beisolated and subjected to DNA sequencing to decode the sequences of thedisplayed antibodies. Through the use of phase display and other methodsknown in the art, high affinity designer antibodies for exosomes can begenerated.

Bead-based assays can also be used to identify novel binding agents toisolate exosomes. A test antibody or peptide can be conjugated to aparticle. For example, a bead can be conjugated to an antibody orpeptide and used to detect and quantify the proteins expressed on thesurface of a population of exosomes in order to discover andspecifically select for novel antibodies that can target exosomes fromspecific tissue or tumor types. Any molecule of organic origin can besuccessfully conjugated to a polystyrene bead through use of acommercially available kit according to manufacturer's instructions.Each bead set can be colored a certain detectable wavelength and eachcan be linked to a known antibody or peptide which can be used tospecifically measure which beads are linked to exosomal proteinsmatching the epitope of previously conjugated antibodies or peptides.The beads can be dyed with discrete fluorescence intensities such thateach bead with a different intensity has a different binding agent asdescribed above.

For example, a purified exosome preparation can be diluted in assaybuffer to an appropriate concentration according to empiricallydetermined dynamic range of assay. A sufficient volume of coupled beadscan be prepared and approximately 1 μl of the antibody-coupled beads canbe aliqouted into a well and adjusted to a final volume of approximately50 μl. Once the antibody-conjugated beads have been added to a vacuumcompatible plate, the beads can be washed to ensure proper bindingconditions: An appropriate volume of exosomal preparation can then beadded to each well being tested and the mixture incubated, such as for15-18 hours. A sufficient volume of detection antibodies using detectionantibody diluent solution can be prepared and incubated with the mixturefor 1 hour or for as long as necessary. The beads can then be washedbefore the addition of detection antibody (biotin expressing) mixturecomposed of streptavidin phycoereythin. The beads can then be washed andvacuum aspirated several times before analysis on a suspension arraysystem using software provided with an instrument. The identity ofantigens that can be used to selectively extract the exosomes can thenbe elucidated from the analysis.

Assays using imaging systems can be utilized to detect and quantifyproteins expressed on the surface of an exosome in order to discover andspecifically select for and enrich exosomes from specific tissue ortumor types. Antibodies, peptides or cells conjugated to multiple wellmultiplex carbon coated plates can be used. Simultaneous measurement ofmany analytes in a well can be achieved through the use of captureantibodies arrayed on the patterned carbon working surface. Analytes canthen be detected with antibodies labeled with reagents in electrodewells with an enhanced electro-chemiluminescent plate. Any molecule oforganic origin can be successfully conjugated to the carbon coatedplate. Proteins expressed on the surface of exosomes can be identifiedfrom this assay and can be used as targets to specifically select forand enrich exosomes from specific tissue or tumor types.

The binding agent can also be a novel aptamer. An aptamer for a targetcan be identified using systematic evolution of ligands by exponentialenrichment (SELEX) (Tuerk & Gold, Science 249:505-510, 1990; Ellington &Szostak, Nature 346:818-822, 1990), such as described in U.S. Pat. No.5,270,163. A library of nucleic acids can be contacted with a targetexosome, and those nucleic acids specifically bound to the target arepartitioned from the remainder of nucleic acids in the library which donot specifically bind the target. The partitioned nucleic acids areamplified to yield a ligand-enriched pool. Multiple cycles of binding,partitioning, and amplifying (i.e., selection) result in identificationof one or more aptamers with the desired activity. Another method foridentifying an aptamer to isolate exosomes is described in U.S. Pat. No.6,376,19, which describes increasing or decreasing frequency of nucleicacids in a library by their binding to a chemically synthesized peptide.Modified methods, such as Laser SELEX or deSELEX as described in U.S.Patent Publication No. 20090264508 can also be used.

Microfluidics

The methods for isolating or identifying exosomes can be used incombination with microfluidic devices. The methods of isolating exosomesdisclosed herein can be performed using microfluidic devices.Microfluidic devices, which may also be referred to as “lab-on-a-chip”systems, biomedical micro-electro-mechanical systems (bioMEMs), ormulticomponent integrated systems, can be used for isolating, andanalyzing, exosomes. Such systems miniaturize and compartmentalizeprocesses that allow for binding of exosomes, detection of exosomalbiomarkers, and other processes.

A microfluidic device can also be used for isolation of an exosomethrough size differential or affinity selection. For example, amicrofluidic device can use one more channels for isolating an exosomefrom a biological sample based on size, or by using one or more bindingagents for isolating an exosome, from a biological sample. A biologicalsample can be introduced into one or more microfluidic channels, whichselectively allows the passage of exosomes. The selection can be basedon a property of the exosomes, for example, size, shape, deformability,biomarker profile, or bio-signature.

Alternatively, a heterogeneous population of exosomes can be introducedinto a microfluidic device, and one or more different homogeneouspopulations of exosomes can be obtained. For example, different channelscan have different size selections or binding agents to select fordifferent exosome populations. Thus, a microfluidic device can isolate aplurality of exosomes, wherein at least a subset of the plurality ofexosomes comprises a different bio-signature from another subset of saidplurality of exosomes. For example, the microfluidic device can isolateat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, or 100 different subsets of exosomes, wherein each subset ofexosomes comprises a different bio-signature.

In some embodiments, the microfluidic device can comprise one or morechannels that permit further enrichment or selection of exosomes. Apopulation of exosomes that has been enriched after passage through afirst channel can be introduced into a second channel, which allows thepassage of the desired exosome population to be further enriched, suchas through binding agents present in the second channel.

Array-based assays and bead-based assays can be used with microfluidicdevice. For example, the binding agent can be coupled to beads and thebinding reaction between the beads and exosomes can be performed in amicrofluidic device. Multiplexing can also be performed using amicrofluidic device. Different compartments can comprise differentbinding agents for different populations of exosomes, where eachpopulation is of a different cell-of-origin specific exosome populationor each population has a different bio-signature. The hybridizationreaction between the microspheres and exosomes can be performed in amicrofluidic device and the reaction mixture can be delivered to adetection device. The detection device, such as a dual or multiple laserdetection system can be part of the microfluidic system and can use alaser to identify each bead or microsphere by its color-coding, andanother laser can detect the hybridization signal associated with eachbead.

Examples of microfluidic devices that may be used, or adapted for usewith exosomes, include but are not limited to those described in U.S.Pat. Nos. 7,591,936, 7,581,429, 7,579,136, 7,575,722, 7,568,399,7,552,741, 7,544,506, 7,541,578, 7,518,726, 7,488,596, 7,485,214,7,467,928, 7,452,713, 7,452,509, 7,449,096, 7,431,887, 7,422,725,7,422,669, 7,419,822, 7,419,639, 7,413,709, 7,411,184, 7,402,229,7,390,463, 7,381,471, 7,357,864, 7,351,592, 7,351,380, 7,338,637,7,329,391, 7,323,140, 7,261,824, 7,258,837, 7,253,003, 7,238,324,7,238,255, 7,233,865, 7,229,538, 7,201,881, 7,195,986, 7,189,581,7,189,580, 7,189,368, 7,141,978, 7,138,062, 7,135,147, 7,125,711,7,118,910, and 7,118,661.

Cell-of-Origin and Disease-Specific Exosomes

The bindings agents disclosed herein can be used to isolate aheterogeneous population of exosomes from a sample or can be used toisolate or identify a homogeneous population of exosomes, such ascell-of-origin specific exosomes or exosomes with specificbio-signatures. A homogeneous population of exosomes, such ascell-of-origin specific exosomes, can be analyzed and used tocharacterize a phenotype for a subject. Cell-of-origin specific exosomesare exosomes derived from specific cell types, which can include, butare not limited to, cells of a specific tissue, cells from a specifictumor of interest or a diseased tissue of interest, circulating tumorcells, or cells of maternal or fetal origin. The exosomes may be derivedfrom tumor cells or lung, pancreas, stomach, intestine, bladder, kidney,ovary, testis, skin, colorectal, breast, prostate, brain, esophagus,liver, placenta, or fetal cells. The isolated exosomes can also be froma particular sample type, such as urinary exosomes.

Cell-of-origin specific exosomes from a biological sample can beisolated using one or more binding agents that are specific to acell-of-origin. Exosomes for analysis of a disease or condition can beisolated using one or more binding agents specific for biomarkers forthat disease or condition.

The exosomes can be concentrated prior to isolation of cell-of-originspecific exosomes, such as through centrifugation, chromatography, orfiltration, as described above, to produce a heterogeneous population ofexosomes prior to isolation of cell-of-origin specific exosomes.Alternatively, the exosomes are not concentrated, or the biologicalsample is not enriched for exosomes, prior to isolation ofcell-of-origin exosomes.

FIG. 61 illustrates a flowchart which depicts one method 100 forisolating or identifying cell-of-origin specific exosomes. First, abiological sample is obtained from a subject in step 102. The sample canbe obtained from a third party or from the same party performing theanalysis. Next, cell-of-origin specific exosomes are isolated from thebiological sample in step 104. The isolated cell-of-origin specificexosomes are then analyzed in step 106 and a biomarker or bio-signaturefor a particular phenotype is identified in step 108. The method may beused for a number of phenotypes. In some embodiments, prior to step 104,exosomes are concentrated or isolated from a biological sample toproduct a heterogeneous population of exosomes. For example,heterogeneous population of exosomes may be isolated usingcentrifugation, chromatography, filtration, or other methods asdescribed above, prior to use of one or more binding agents specific forisolating or identifying exosomes derived from specific cell types, orcell-of-origin specific exosomes.

Cell-of-origin specific exosomes can be isolated from a biologicalsample of a subject by employing one or more binding agents that bindwith high specificity to the cell-of-origin specific exosomes. In someinstances, a single binding agent can be employed to isolatecell-of-origin specific exosomes. In other instances, a combination ofbinding agents may be employed to isolate cell-of-origin specificexosomes. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 50, 75, or 100 different binding agentsmay be used to isolate cell-of-origin exosomes. Therefore, an exosomepopulation (e.g., exosomes having the same binding agent profile) can beidentified by utilizing a single or a plurality of binding agents.

One or more binding agents can be selected based on their specificityfor a target antigen(s) that is specific to a cell-of-origin, tumor ordisease. Non-limiting examples of antigens which may be used singularly,or in combination, to isolate a cell-of-origin specific exosome, diseasespecific exosome, or tumor specific exosome is shown in FIG. 1 and arealso described below. The antigen may be membrane bound antigens whichare accessible to binding agents. The antigen can also be a biomarkerfor the phenotype.

Breast Cancer

An exosome derived from a breast cancer cell can be isolated using abinding agent (e.g., antibody), that is specific for an antigen that isassociated with a cell of breast cancer origin (e.g., cells of glandularor stromal origin). An exosome derived from a breast cancer cell can beisolated using an antigen including, but not limited to, BCA-225, hsp70,MART1, ER, VEGFA, Class III b-tubulin, HER2/neu (for Her2+BC), GPR30,ErbB4 (JM) isoform, MPR8, MISIIR, fragments thereof, any combinationthereof, or any combination of antigens that are specific for a breastcancer cell.

Ovarian Cancer

An exosome derived from an ovarian cancer cell can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor a cell of ovarian cancer origin including, but not limited to,CA125, VEGFR2, HER2, MISIIR, VEGFA, CD24, fragments thereof, anycombination thereof, or any combination of antigens that is specific foran ovarian cancer cell.

Lung Cancer

An exosome derived from a lung cancer cell can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor a cell of lung cancer origin including, but not limited to,CYFRA21-1, TPA-M, TPS, CEA, SCC-Ag, XAGE-1b, HLA Class 1, TA-MUC1, KRAS,hENT1, kinin B1 receptor, kinin B2 receptor, TSC403, HTI56, DC-LAMP,fragments thereof, any combination thereof, or any combination ofantigens that is specific for a lung cancer cell.

Colon Cancer

An exosome derived from a colon cancer cell can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor a cell of colon cancer origin including, but not limited to, CEA,MUC2, GPA33, CEACAM5, ENFB1, CCSA-3, CCSA-4, ADAM10, CD44, NG2, ephrinB1, plakoglobin, galectin 4, RACK1, tetraspanin-8, FASL, A33, CEA, EGFR,dipeptidase 1, PTEN, Na(+)-dependent glucose transporter,UDP-glucuronosyltransferase 1A, fragments thereof, any combinationthereof, or any combination of antigens that is specific for a coloncancer cell.

Prostate Cancer

An exosome derived from a prostate cancer cell can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor a cell of prostate cancer origin including, but not limited to, PSA,TMPRSS2, FASLG, TNFSF10, PSMA, NGEP, Il-7RI, CSCR4, CysLT1R, TRPM8,Kv1.3, TRPV6, TRPM8, PSGR, MISIIR, galectin-3, PCA3, TMPRSS2:ERG,fragments thereof, any combination thereof, or any combination ofantigens that is specific for a prostate cancer cell.

Brain Cancer

An exosome derived from brain cancer cell can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor a cell of brain cancer origin including, but not limited to, PRMT8,BDNF, EGFR, DPPX, Elk, Densin-180, BAI2, BAI3, fragments thereof, anycombination thereof, or any combination of antigens that is specific fora brain cancer cell.

Blood Cancer

An exosome derived from a hematological malignancy cell can be isolatedusing an antibody, or any other binding agent, for an antigen that isspecific for a cell of hematological malignancy origin including, butnot limited to, CD44, CD58, CD31, CD11a, CD49d, GARP, BTS, Raftlin,fragments thereof, any combination thereof, or any combination ofantigens that is specific for a hematological malignancy cell.

Melanoma

An exosome derived from a melanoma cell can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor a cell of melanoma origin including, but not limited to, DUSP1,TYRP1, SILV, MLANA, MCAM, CD63, Alix, hsp70, meosin, p120 catenin, PGRL,syntaxin binding protein 1 &2, caveolin, fragments thereof, anycombination thereof, or any combination of antigens that is specific fora melanoma cell.

Liver Cancer

An exosome derived from a hepatocellular carcinoma cell can be isolatedusing an antibody, or any other binding agent, for an antigen that isspecific for a cell of hepatocellular carcinoma origin including, butnot limited to, HBxAg, HBsAg, NLT, fragments thereof, any combinationthereof, or any combination of antigens that is specific for ahepatocellular carcinoma cell.

Cervical Cancer

An exosome derived from a cervical cancer cell can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor a cell of cervical cancer origin including, but not limited to,MCT-1, MCT-2, MCT-4, fragments thereof, any combination thereof, or anycombination of antigens that is specific for a cervical cancer cell.

Endometrial Cancer

An exosome derived from an endometrial cancer cell can be isolated usingan antibody, or any other binding agent, for an antigen that is specificfor a cell of endometrial cancer origin including, but not limited to,Alpha V Beta 6 integrin, fragments thereof, any combination thereof, orany combination of antigens that is specific for an endometrial cancercell.

Psoriasis

An exosome for characterizing psoriasis can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor psoriasis including, but not limited to, flt-1, VPF receptors, kdr,fragments thereof, any combination thereof, or any combination ofantigens that is specific to psoriasis.

Autoimmune Disease

An exosome for characterizing an autoimmune disease can be isolatedusing an antibody, or any other binding agent, for an antigen that isspecific for an autoimmune disease including, but not limited to, Tim-2,fragments thereof, or any combination of antigens that is specific to anautoimmune disease.

Irritable Bowel Disease

An exosome for characterizing irritable bowel disease (IBD) or syndrome(IBS) can be isolated using an antibody, or any other binding agent, foran antigen that is specific for IBD or IBS including, but not limitedto, IL-16, IL-1beta, IL-12, TNF-alpha, interferon-gamma, IL-6, Rantes,11-12, MCP-1, 5HT, fragments thereof, or any combination of antigensthat is specific to IBD or IBS.

Diabetes

An exosome derived from a pancreatic cell can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor a cell of pancreatic origin including, but not limited to, IL-6,CRP, RBP4, fragments thereof, any combination thereof, or anycombination of antigens that is specific for a pancreatic cell.

Barrett's Esophagus

An exosome for characterizing Barrett's Esophagus can be isolated usingan antibody, or any other binding agent, for an antigen that is specificfor Barrett's Esophagus including, but not limited to, p53, MUC1, MUC6,fragments thereof, any combination thereof, or any combination ofantigens that is specific to Barrett's Esophagus.

Fibromyalgia

An exosome for characterizing fibromyalgia can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor fibromyalgia including, but not limited to, neopterin, gp130,fragments thereof, any combination thereof, or any combination ofantigens that is specific to fibromyalgia.

Prostatic Hyperplasia

An exosome derived from a benign prostatic hyperplasia (BPH) cell can beisolated using an antibody, or any other binding agent, for an antigenthat is specific for a cell of BPH origin including, but not limited to,KIA1, intact fibronectin, fragments thereof, any combination thereof, orany combination of antigens that is specific for a BPH cell.

Multiple Sclerosis

An exosome for characterizing multiple sclerosis (MS) can be isolatedusing an antibody, or any other binding agent, for an antigen that isspecific for MS including, but not limited to, B7, B7-2, CD-95 (fas),Apo-1/Fas, fragments thereof, any combination thereof, or anycombination of antigens that is specific to MS.

Parkinson's Disease

An exosome for characterizing Parkinson's disease can be isolated usingan antibody, or any other binding agent, for an antigen that is specificfor Parkinson's disease including, but not limited to, PARK2,ceruloplasmin, VDBP, tau, DJ-1, fragments thereof, any combinationthereof, or any combination of antigens that is specific to Parkinson'sdisease.

Rheumatic Disease

An exosome for characterizing rheumatic disease can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor rheumatic disease including, but not limited to, Citrulinated fibrina-chain, CD5 antigen-like fibrinogen fragment D, CD5 antigen-likefibrinogen fragment B, TNF alpha, fragments thereof, any combinationthereof, or any combination of antigens that is specific to rheumaticdisease.

Alzheimer's Disease

An exosome derived from a neuron of a patient suffering from Alzheimer'sdisease can be further isolated using an antibody, or any other bindingagent, for an antigen that including, but not limited to, APP695, APP751or APP770, BACE1, cystatin C, amyloid β, T-tau, complement factor H oralpha-2-macroglobulin, fragments thereof, any combination thereof, orany combination of antigens that are specific for Alzheimer's.

Head and Neck Cancer

An exosome derived from a head and neck cancer cell can be isolatedusing an antibody, or any other binding agent, for an antigen that isspecific for a cell of head and neck cancer origin including, but notlimited to, EGFR, EphB4 or Ephrin B2, fragments thereof, any combinationthereof, or any combination of antigens that is specific for a head andneck cancer cell.

Gastrointestinal Stromal Tumor

An exosome derived from a gastrointestinal stromal tumor (GIST) cell canbe isolated using an antibody, or any other binding agent, for anantigen that is specific for a cell of GIST origin including, but notlimited to, c-kit PDGFRA, NHE-3, fragments thereof, any combinationthereof, or any combination of antigens that is specific for a GISTcell.

Renal Cell Carcinoma

An exosome derived from a renal cell carcinomas (RCC) cell can beisolated using an antibody, or any other binding agent, for an antigenthat is specific for a cell of RCC origin including, but not limited to,c PDGFRA, VEGF, HIF 1 alpha, fragments thereof, any combination thereof,or any combination of antigens that is specific for a RCC cell.

Schizophrenia

An exosome for characterizing schizophrenia can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor schizophrenia including, but not limited to, ATP5B, ATP5H, ATP6V1B,DNM1, fragments thereof, any combination thereof, or any combination ofantigens that is specific to schizophrenia.

Peripheral Neuropathic Pain

An exosome derived from a nerve cell of a patient suffering fromperipheral neuropathic pain can be isolated using an antibody, or anyother binding agent, for an antigen that is specific for peripheralneuropathic pain including, but not limited to, OX42, ED9, fragmentsthereof, any combination thereof, or any combination of antigens that isspecific for peripheral neuropathic pain.

Chronic Neuropathic Pain

An exosome derived from a nerve cell of a patient suffering from chronicneuropathic pain can be isolated using an antibody, or any other bindingagent, for an antigen that is specific for chronic neuropathic painincluding, but not limited to, chemokine receptor (CCR2/4), fragmentsthereof, or any combination of antigens that is specific for chronicneuropathic pain.

Prion Disease

An exosome derived from a cell of a patient suffering from prion diseasecan be isolated using an antibody, or any other binding agent, for anantigen that is specific for prion disease including, but not limitedto, PrPSc, 14-3-3 zeta, S-100, AQP4, fragments thereof, or anycombination of antigens that is specific for prion disease.

Stroke

An exosome for characterizing stroke can be isolated using an antibody,or any other binding agent, for an antigen that is specific for strokeincluding, but not limited to, S-100, neuron specific enolase, PARK7,NDKA, ApoC-I, ApoC-III, SAA or AT-III fragment, Lp-PLA2, hs-CRP,fragments thereof, any combination thereof, or any combination ofantigens that is specific to stroke.

Cardiovascular Disease

An exosome for characterizing a cardiovascular disease can be isolatedusing an antibody, or any other binding agent, for an antigen that isspecific for a cardiovascular disease including, but not limited to,FATP6, fragments thereof, or any combination of antigens that isspecific to a cardiovascular disease or cardiac cell.

Esophageal Cancer

An exosome derived from an esophageal cancer cell can be isolated usingan antibody, or any other binding agent, for an antigen that is specificfor a cell of esophageal cancer origin including, but not limited to,CaSR, fragments thereof, or any combination of antigens that is specificfor an esophageal cancer cell.

Tuberculosis

An exosome for characterizing tuberculosis (TB) can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor TB including, but not limited to, antigen 60, HSP,Lipoarabinomannan, Sulfolipid, antigen of acylated trehalose family,DAT, TAT, Trehalose 6,6-dimycolate (cord-factor) antigen, fragmentsthereof, any combination thereof, or any combination of antigens that isspecific to TB.

HIV

An exosome for characterizing HIV can be isolated using an antibody, orany other binding agent, for an antigen that is specific for HIVincluding, but not limited to, gp41, gp120, fragments thereof, anycombination thereof, or any combination of antigens that is specific toHIV.

Autism

An exosome for characterizing autism can be isolated using an antibody,or any other binding agent, for an antigen that is specific for autismincluding, but not limited to, VIP, PACAP, CGRP, NT3, fragments thereof,any combination thereof, or any combination of antigens that is specificto autism.

Asthma

An exosome for characterizing asthma can be isolated using an antibody,or any other binding agent, for an antigen that is specific for asthmaincluding, but not limited to, YKL-40, S-nitrosothiols, SSCA2, PAI,amphiregulin, periostin, fragments thereof, any combination thereof, orany combination of antigens that is specific to asthma.

Lupus

An exosome for characterizing lupus can be isolated using an antibody,or any other binding agent, for an antigen that is specific for lupusincluding, but not limited to, TNFR, fragments thereof, or anycombination of antigens that is specific to lupus.

Cirrhosis

An exosome for characterizing cirrhosis can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor cirrhosis including, but not limited to, NLT, HBsAg, fragmentsthereof, any combination thereof, or any combination of antigens that isspecific to cirrhosis.

Influenza

An exosome for characterizing influenza can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor influenza including, but not limited to, hemagglutinin,neurominidase, fragments thereof, any combination thereof, or anycombination of antigens that is specific to influenza.

Vulnerable Plaque

An exosome for characterizing vulnerable plaque can be isolated using anantibody, or any other binding agent, for an antigen that is specificfor vulnerable plaque including, but not limited to, Alpha v. Beta 3integrin, MMP9, fragments thereof, any combination thereof, or anycombination of antigens that is specific to vulnerable plaque.

A cell-of-origin specific exosome may be isolated using novel bindingagents, using methods as described above. Furthermore, a cell-of-originspecific exosome can also be isolated from a biological sample usingisolation methods based on cellular binding partners or binding agentsof such exosomes. Such cellular binding partners can include but are notlimited to peptides, proteins, RNA, DNA, apatmers, cells orserum-associated proteins that only bind to such exosomes when one ormore specific biomarkers are present. Isolation of a cell-of-originspecific exosome can be carried out with a single binding partner orbinding agent, or a combination of binding partners or binding agentswhose singular application or combined application results incell-of-origin specific isolation. Non-limiting examples of such bindingagents are provided in FIG. 2. For example, an exosome forcharacterizing breast cancer can be isolated with one or more bindingagents including, but not limited to, estrogen, progesterone, Herceptin(Trastuzumab), CCND1, MYC PNA, IGF-1 PNA, MYC PNA, SC4 aptamer (Ku),AII-7 aptamer (ERB2), Galectin-3, mucin-type O-glycans, L-PHA,Galectin-9, or any combination thereof.

A binding agent may also be used for isolating the cell-of-originspecific exosome based on i) the presence of antigens specific forcell-of-origin specific exosomes cells, ii) the absence of markersspecific for cell-of-origin specific exosomes, or iii) expression levelsof biomarkers specific for cell-of-origin specific exosomes. Aheterogeneous population of exosomes is applied to a surface coated withspecific binding agents designed to rule out or identify thecell-of-origin characteristics of the exosomes. Various binding agents,such as antibodies, can be arrayed on a solid surface or substrate andthe heterogeneous population of exosomes is allowed to contact the solidsurface or substrate for a sufficient time to allow interactions to takeplace. Specific binding or non-binding to given antibody locations onthe array surface or substrate can then serve to identify antigenspecific characteristics of the exosome population that are specific toa given cell-of-origin.

A cell-of-origin specific exosome can be enriched or isolated using oneor more binding agents using a magnetic capture method, fluorescenceactivated cell sorting or laser cytometry as described above. Magneticcapture methods can include, but are not limited to, the use ofmagnetically activated cell sorter (MACS) microbeads or magneticcolumns. Examples of immunoaffinity and magnetic particle methods thatcan be used is described in U.S. Pat. No. 4,551,435, 4,795,698,4,925,788, 5,108,933, 5,186,827, 5,200,084 or 5,158,871. Acell-of-origin specific exosome can also be isolated following thegeneral methods described in U.S. Pat. No. 7,399,632, by usingcombination of antigens specific to an exosome.

Any other method for isolating or otherwise enriching the cell-of-originspecific exosomes with respect to a biological sample may also be usedin combination with the present invention. For example, size exclusionchromatography such as gel permeation columns, centrifugation or densitygradient centrifugation, and filtration methods can be used incombination with the antigen selection methods described herein. Thecell-of-origin specific exosomes may also be isolated following themethods described in Koga et al., Anticancer Research, 25:3703-3708(2005), Taylor et al., Gynecologic Oncology, 110:13-21 (2008), Nanjee etal., Clin Chem, 2000; 46:207-223 or U.S. Pat. No. 7,232,653.

Exosome Assessment

A phenotype can be characterized for a subject by analyzing a biologicalsample from the subject and determining the level, amount, orconcentration of one or more populations of exosomes in the sample. Anexosome can be purified or concentrated prior to determining the amountof exosomes. Alternatively, the amount of exosomes can be directlyassayed from a sample, without prior purification or concentration. Theexosomes can be cell-of-origin specific exosomes or exosomes with aspecific biomarker or combination of biomarkers. The amount of exosomescan be used to characterize a phenotype, such as a diagnosis, theranosisor prognosis of a condition or disease. The amount may be used todetermine a physiological or biological state, such as pregnancy or thestage of pregnancy. The amount of exosomes can also be used to determinetreatment efficacy, stage of a disease or condition, or progression of adisease or condition. For example, the amount of exosomes can beproportional to an increase in disease stage or progression.

The exosomes can be evaluated by comparing the level of exosomes with areference level or value of exosomes. The reference value can beparticular to physical or temporal endpoint. For example, the referencevalue can be from the same subject from whom a sample is assessed for anexosome, or the reference value can be from a representative populationof samples (e.g., samples from normal subjects not exhibiting a symptomof disease). Therefore, a reference value can provide a thresholdmeasurement which is compared to a subject sample's readout for one ormore exosome populations assayed in a given sample. Such referencevalues may be set according to data pooled from groups of samplecorresponding to a particular cohort, including but not limited to age(e.g., newborns, infants, adolescents, young, middle-aged adults,seniors and adults of varied ages), racial/ethnic groups, normal versusdiseased subjects, smoker v. non-smoker, subject receiving therapyversus untreated subject, different time points of treatment for aparticular individual or group of subjects similarly diagnosed ortreated or combinations thereof.

A reference value may be based on samples assessed from the same subjectso to provide individualized tracking. Frequent testing of a patient mayprovide better comparisons to the reference values previouslyestablished for a particular patient and would allow a physician to moreaccurately assess the patient's disease stage or progression, and toinform a better decision for treatment. The reduced intraindividualvariance of exosomes levels would allow a more specific andindividualized threshold to be defined for the patient. Temporalintrasubject variation allows each individual to serve as a longitudinalcontrol for optimum analysis of disease or physiological state.

Reference values can be established for unaffected individuals (ofvarying ages, ethnic backgrounds and sexes) without a particularphenotype by determining the amount of exosomes in an unaffectedindividual. For example, a reference value for a reference populationcan be utilized as a baseline for detection of one or more exosomepopulations in a test subject. If a sample from a subject has a level orvalue that is similar to the reference, the subject can be identified tonot have the disease, or of having a low likelihood of developing adisease.

Alternatively, reference values or levels can be established forindividuals with a particular phenotype by determining the amount of oneor more populations of exosomes in an individual with the phenotype. Inaddition, an index of values can be generated for a particularphenotype. For example, different disease stages can have differentvalues, such as obtained from individuals with the different diseasestages. A subject's value can be compared to the index and a diagnosisor prognosis of the disease can be determined, such as the disease stageor progression. In other embodiments, an index of values is generatedfor therapeutic efficacies. For example, the level of exosomes ofindividuals with a particular disease can be generated and noted whattreatments were effective for the individual. The levels can be used togenerate values of which is a subject's value is compared, and atreatment or therapy can be selected for the individual.

For example, a reference value can be determined for individualsunaffected with a particular cancer, by isolating exosomes with anantigen that specifically targets for the particular cancer. Forexample, individuals with varying stages of colorectal cancer andnoncancerous polyps can be surveyed using the same techniques describedfor unaffected individuals and the levels of circulating exosomes foreach group defined as means±standard deviations from at least twoseparate experiments performed in triplicate. Comparisons between thesegroups can be made using statistical applications such as one-way ANOVA,followed by Tukey's multiple comparisons post-test comparing eachpopulation.

Reference values can also be established for disease recurrencemonitoring (or exacerbation phase in MS), or for therapeutic responsemonitoring.

The values can be a quantitative or qualitative value. The values can bea direct measurement of the level of exosomes (example, mass pervolume), or an indirect measure, such as the amount of a specificexosomal marker. The values can be a quantitative, such as a numericalvalue. In other embodiments, the value is qualitiative, such as noexosomes, low level of exosomes, medium level, or high level ofexosomes, or variations thereof.

The reference values can be stored in a database and used as a referencefor the diagnosis, prognosis, or theranosis of a disease or conditionbased on the level or amount of exosomes, such as total amount ofexosomes, or the amount of a specific population of exosomes, such ascell-of-origin specific exosomes or exosomes with one or more specificbiomarkers.

Exosome levels may be characterized using mass spectrometry or flowcytometry. Analysis may also be carried out on exosomes byimmunocytochemical staining, Western blotting, electrophoresis,chromatography or x-ray crystallography in accordance with procedureswell known in the art. Exosomes may be characterized and quantitativelymeasured using flow cytometry as described in Clayton et al., Journal ofImmunological Methods 2001; 163-174, which is herein incorporated byreference in its entirety. Exosome levels may be determined usingbinding agents as described above. For example, a binding agent toexosomes can be labeled and the label detected and used to determine theamount of exosomes in a sample. The binding agent can be bound to asubstrate, such as arrays or particles, such as described above.Alternatively, the exosomes may be labeled directly.

Electrophoretic tags or eTags can also be used to determine the amountof exosomes. eTags are small fluorescent molecules linked to nucleicacids or antibodies and are designed to bind one specific nucleic acidsequence or protein, respectively. After the eTag binds its target, anenzyme is used to cleave the bound eTag from the target. The signalgenerated from the released eTag, called a “reporter,” is proportionalto the amount of target nucleic acid or protein in the sample. The eTagreporters can be identified by capillary electrophoresis. The uniquecharge-to-mass ratio of each eTag reporter—that is, its electricalcharge divided by its molecular weight—makes it show up as a specificpeak on the capillary electrophoresis readout. Thus by targeting aspecific biomarker of an exosome with an eTag, the amount or level ofexosomes can be determined.

The exosome levels can determined from a heterogeneous population ofexosomes, such as the total population of exosomes in a sample.Alternatively, the exosomes level is determined from a homogenouspopulation, or substantially homogenous population of exosomes, such asthe level of specific cell-of-origin exosomes, such as exosomes fromprostate cancer cells. In yet other embodiments, the level is determinedfor exosomes with a particular biomarker or combination of biomarkers,such as a biomarker specific for prostate cancer. Determining the levelof exosome can be performed in conjunction with determining thebiomarker or combination of biomarkers of an exosome. Alternatively,determining the amount of exosome may be performed prior to orsubsequent to determining the biomarker or combination of biomarkers ofthe exosomes.

Determining the amount of exosomes can be assayed in a multiplexedmanner. For example, determining the amount of more than one populationof exosomes, such as different cell-of-origin specific exosomes orexosomes with different biomarkers or combination of biomarkers, can beperformed, such as those disclosed herein.

Specificity and Sensitivity

The level of exosomes as determined using one or more processesdisclosed herein can be used to characterize a phenotype with increasedsensitivity and the specificity. The sensitivity can be determined by:(number of true positives)/(number of true positives+number of falsenegatives). The specificity can be determined by: (number of truenegatives)/(number of true negatives+number of false positives).

The level of exosomes as determined using one or more processesdisclosed herein can be used to characterize a phenotype with at least60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70% sensitivity, such as withat least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,or 87% sensitivity. For example, the phenotype can be characterized withat least 87.1, 87.2, 87.3, 87.4, 87.5, 87.6, 87.7, 87.8, 87.9, 88.0, or89% sensitivity, such as with at least 90% sensitivity. The phenotypecan be characterized with at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or100% sensitivity.

The phenotype of a subject can also be characterized with at least 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, or 97% specificity, such as with atleast 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0,98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% specificity.

The phenotype can also be characterized with at least 70% sensitivityand at least 80, 90, 95, 99, or 100% specificity; at least 75%sensitivity and at least 80, 90, 95, 99, or 100% specificity; at least80% sensitivity and at least 80, 85, 90, 95, 99, or 100% specificity; atleast 85% sensitivity and at least 80, 85, 90, 95, 99, or 100%specificity; at least 86% sensitivity and at least 80, 85, 90, 95, 99,or 100% specificity; at least 87% sensitivity and at least 80, 85, 90,95, 99, or 100% specificity; at least 88% sensitivity and at least 80,85, 90, 95, 99, or 100% specificity; at least 89% sensitivity and atleast 80, 85, 90, 95, 99, or 100% specificity; at least 90% sensitivityand at least 80, 85, 90, 95, 99, or 100% specificity; at least 95%sensitivity and at least 80, 85, 90, 95, 99, or 100% specificity; atleast 99% sensitivity and at least 80, 85, 90, 95, 99, or 100%specificity; or at least 100% sensitivity and at least 80, 85, 90, 95,99, or 100% specificity.

Furthermore, the confidence level for determining the specificity,sensitivity, or both, may be with at least 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% confidence.

Bio-Signatures

A bio-signature of an exosome from a subject can be used to characterizea phenotype. A bio-signature can reflect the particular antigens orbiomarkers that are present on an exosome. In addition, a bio-signaturecan also reflect one or more biomarkers that are carried in an exosome.Alternatively, a bio-signature can comprise a combination of one or moreantigens or biomarkers that are present on an exosome with one or morebiomarkers that are detected in the exosome.

The exosome can be purified or concentrated prior to determining thebio-signature of the exosome. Alternatively, the bio-signature of theexosome can be directly assayed from a sample, without priorpurification or concentration. An exosome can also be isolated prior toassaying. For example, a cell-of-origin specific exosome can be isolatedand its bio-signature determined. The bio-signature is used to determinea diagnosis, prognosis, or theranosis of a disease or condition.Therefore, a bio-signature can also be used to determine treatmentefficacy, stage of a disease or condition, or progression of a diseaseor condition. Furthermore, a bio-signature may be used to determine aphysiological state, such as pregnancy.

An exosomal characteristic in and of itself can be assessed to determinea bio-signature. The exosomal characteristic can be used to diagnose,detect or determine a disease stage or progression, the therapeuticimplications of a disease or condition, or characterize a physiologicalstate. An exosomal characteristic can include, but is not limited to,the level or amount of exosomes, temporal evaluation of the variation inexosomal half-life, circulating exosomal half-life or exosomal metabolichalf-life, or the activity of an exosome.

In addition, a bio-signature can also correspond to an expression level,presence, absence, mutation, variant, copy number variation, truncation,duplication, modification, or molecular association of one or morebiomarkers. A biomarker may be any exosomal component and can form itsown signature. For example, the biomarker may be the RNA content of theexosome, such that the RNA signature includes one or more RNA species,such as, but not limited to, mRNA, miRNA, snoRNA, snRNA, rRNAs, tRNAs,siRNA, hnRNA, shRNA, or a combination thereof. Therefore, an exosome canbe assayed to determine a RNA signature.

Other biomarkers include, but are not limited to, one or more proteinsor peptides (e.g., providing a protein signature), nucleic acids (e.g.RNA signature as described, or a DNA signature), lipids (e.g. lipidsignature), or combinations thereof. In some embodiments, thebio-signature can also comprise the type or amount of drug or drugmetabolite present in an exosome (e.g. drug signature), as such drug maybe taken by a subject from which the biological sample is obtained from,resulting in an exosome carrying such drug, or metabolites of such drug.

An RNA signature or DNA signature can also include a mutational,epigenetic modification, or genetic variant analysis of the RNA or DNApresent in the exosome. In addition, a protein signature can include,but is not limited to, the mutation, modification, overexpression,underexpression, presence or absence of antigens, peptides, proteins orcombinations thereof.

A bio-signature of an exosome can comprise one or more miRNA signaturescombined with one or more additional signatures including, but notlimited to, an mRNA signature, DNA signature, protein signature, peptidesignature, antigen signature, or any combination thereof. For example,the bio-signature can comprise one or more miRNA biomarkers with one ormore DNA biomarkers, one or more mRNA biomarkers, one or more snoRNAbiomarkers, one or more protein biomarkers, one or more peptidebiomarkers, one or more antigen biomarkers, one or more antigenbiomarkers, one or more lipid biomarkers, or any combination thereof.

A bio-signature can comprise a combination of one or more antigens orbinding agents (such as ability to bind one or more binding agents),such as listed in FIGS. 1 and 2, respectively. The bio-signature canfurther comprise one or more other biomarkers, such as, but not limitedto, miRNA, DNA (e.g. single stranded DNA, complementary DNA, ornoncoding DNA), or mRNA. For example, the bio-signature of an exosomecan comprise a combination of one or more antigens, such as shown inFIG. 1, one or more binding agents, such as shown in FIG. 2, and one ormore biomarkers for a condition or disease, such as listed in FIGS.3-60. The bio-signature can comprise one or more biomarkers, for examplemiRNA, with one or more antigens specific for a cancer cell (forexample, as shown in FIG. 1).

An exosome can have a bio-signature that is specific to thecell-of-origin and, as such, can be utilized to derive disease-specificor biological state specific diagnostic, prognostic or therapy-relatedbio-signatures representative of the cell-of-origin. An exosome may alsohave a bio-signature that is specific to a given disease orphysiological condition that may be different from the bio-signature ofthe cell-of-origin, but no less important to the diagnosis, prognosis,staging, therapy-related determinations or physiological statecharacterization.

The bio-signature of an exosome, such as a cell-of-origin specificexosome described herein, can be used clinically in making decisionsconcerning treatment modalities, including therapeutic intervention,diagnostic criteria such as disease staging, disease monitoring, anddisease stratification, and surveillance for detection, metastasis orrecurrence or progression of disease. The bio-signature of an exosome,such as an isolated cell-of-origin specific exosome can further be usedclinically to make treatment decisions, including whether to performsurgery or what treatment standards should be utilized along withsurgery (e.g., either pre-surgery or post-surgery).

An exosome bio-signature can also be used in therapy related diagnosticsto provide tests useful to diagnose a disease or choose the correcttreatment regimen, as well as monitor a subject's response. Therapyrelated tests are useful to predict and assess drug response inindividual subjects, i.e., to provide personalized medicine. Therapyrelated tests are also useful to select a subject for treatment who isparticularly likely to benefit from the treatment or to provide an earlyand objective indication of treatment efficacy in an individual subject.For example, a treatment can be altered without the great expense ofdelaying beneficial treatment as well as the great financial cost ofadministering an ineffective drug(s).

Therapy related diagnostics are also useful in clinical diagnosis andmanagement of a variety of diseases and disorders, which include, butare not limited to cardiovascular disease, cancer, infectious diseases,sepsis, neurological diseases, central nervous system related diseases,endovascular related diseases, and autoimmune related diseases or theprediction of drug toxicity, drug resistance or drug response. Therapyrelated tests may be developed in any suitable diagnostic testingformat, which include, but are not limited to, e.g., immunohistochemicaltests, clinical chemistry, immunoassay, cell-based technologies, nucleicacid tests or body imaging methods. Therapy related tests can furtherinclude but are not limited to, testing that aids in the determinationof therapy, testing that monitors for therapeutic toxicity, or responseto therapy testing. For example, a bio-signature can determine whether aparticular disease or condition is resistant to a drug, and therefore, aphysician need not waste valuable time with hit-and-miss treatment.Instead, to obtain early validation of a drug choice or treatmentregimen, a bio-signature is determined for an exosome obtained from asubject, which then determines whether the particular subject's diseasehas the biomarker associated with drug resistance. Therefore, such adetermination enables doctors to devote critical time as well as thepatient's financial resources to effective treatments.

Moreover, an exosome bio-signature may be used to assess whether asubject is afflicted with disease, is at risk for developing disease orto assess the stage or progression of the disease. For example, abio-signature can be used to assess whether a subject has prostatecancer (for example, FIGS. 68, 73) or colon cancer (for example, FIGS.69, 74). Furthermore, a bio-signature can be used to determine a stageof a disease or condition, such as colon cancer (for example, FIGS. 71,72).

Furthermore, determining the amount of exosomes, such a heterogeneouspopulation of exosomes, and the amount of one or more homogeneouspopulation of exosomes, such as a population of exosomes with the samebio-signature, can be used to characterize a phenotype. For example,determination of the total amount of exosomes in a sample (i.e. notcell-type specific) and determining the presence of one or moredifferent cell-of-origin specific exosomes (such as cell-of-originspecific exosomes) can be used to characterize a phenotype. Thresholdvalues, or reference values or amounts can be determined based oncomparisons of normal subjects and subjects with the phenotype ofinterest, as further described below, and criteria based on thethreshold or reference values determined. The different criteria can beused to characterize a phenotype.

For example, one criterion can be based on the amount of a heterogeneouspopulation of exosomes in a sample. If the amount is lower than athreshold value or reference value, the criterion is met. Alternatively,the criterion can be based on whether the amount of exosomes is higherthan a threshold or reference value. Another criterion can be the amountof exosomes with a specific bio-signature or biomarker. If the amount ofexosomes with the specific bio-signature or biomarker is lower, orhigher, than a threshold or reference value, the criterion is met. Acriterion can also be based on the amount of exosomes derived from aparticular cell type. If the amount is lower, or higher, than athreshold or reference value, the criterion is met. Another criterioncan be based on whether the amount of exosomes derived from a cancercell or comprising one or more cancer specific biomarkers. If the amountis lower, or higher, than a threshold or reference value, the criterionis met. A criterion can also be the reliability of the result, such asmeeting a quality control measure or value.

A phenotype for a subject can be characterized based on meeting at least1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 criteria. For example, for thecharacterizing of a cancer, a number of different criteria can beused: 1) if the amount of exosomes in a sample from a subject is higherthan a reference value; 2) if the amount of a cell type (ie. derivedfrom a specific tissue or organ) specific exosomes is higher than areference value; and 3) if the amount of exosomes with one or morecancer specific biomarkers is higher than a reference value, the subjectis diagnosed with a cancer. The method can further include a qualitycontrol measure, such that the results are provided for the subject ifthe samples meet the quality control measure.

A bio-signature can be determined by comparing the amount of exosomes,the structure of an exosome, (using transmission electron microscopy,see for example, Hansen et al., Journal of Biomechanics 31, Supplement1: 134-134(1) (1998), or scanning electron microscopy), or any otherexosomal characteristic. Various combinations of methods and techniquesor analyzing one or more exosomes can be used to determine a phenotypefor a subject.

An exosome characteristic can include, but is not limited to thepresence or absence, copy number, expression level, or activity level ofa biomarker. The presence of a mutation (e.g., mutations which affectactivity of the biomarker, such as substitution, deletion, or insertionmutations), variant, or post-translation modification of a biomarker,such as a protein biomarker, can include, but not be limited to,acylation, acetylation, phosphorylation, ubiquitination, deacetylation,alkylation, methylation, amidation, biotinylation, gamma-carboxylation,glutamylation, glycosylation, glycyation, hydroxylation, covalentattachment of heme moiety, iodination, isoprenylation, lipoylation,prenylation, GPI anchor formation, myristoylation, farnesylation,geranylgeranylation, covalent attachment of nucleotides or derivativesthereof, ADP-ribosylation, flavin attachment, oxidation, palmitoylation,pegylation, covalent attachment of phosphatidylinositol,phosphopantetheinylation, polysialylation, pyroglutamate formation,racemization of proline by prolyl isomerase, tRNA-mediation addition ofamino acids such as arginylation, sulfation, the addition of a sulfategroup to a tyrosine, or selenoylation of the biomarker can also be anexosomal characteristic.

The methods described above can be used to identify an exosomebio-signature that is associated with a disease, condition orphysiological state.

The bio-signature can also be utilized to determine if a subject isafflicted with cancer or is at risk for developing cancer. A subject atrisk of developing cancer can include those who may be predisposed orwho have pre-symptomatic early stage disease.

A bio-signature can also be utilized to provide a diagnostic ortheranostic determination for other diseases including but not limitedto autoimmune diseases, inflammatory bowel diseases, Alzheimer'sdisease, Parkinson's disease, Multiple Sclerosis, sepsis or pancreatitisor any disease, conditions or symptoms listed in FIGS. 3-58.

The bio-signature can also be used to identify a given pregnancy statefrom the peripheral blood, umbilical cord blood, or amniotic fluid (e.g.miRNA signature specific to Downs Syndrome) or adverse pregnancy outcomesuch as pre-eclampsia, pre-term birth, premature rupture of membranes,intrauterine growth restriction or recurrent pregnancy loss. Thebio-signature can also be used to indicate the health of the mother, thefetus at all developmental stages, the pre-implantation embryo or anewborn.

A bio-signature can be utilized for pre-symptomatic diagnosis.Furthermore, the bio-signature can be utilized to detect disease,determine disease stage or progression, determine the recurrence ofdisease, identify treatment protocols, determine efficacy of treatmentprotocols or evaluate the physiological status of individuals related toage and environmental exposure.

Monitoring the bio-signature of an exosome can also be used to identifytoxic exposures in a subject including, but not limited to, situationsof early exposure or exposure to an unknown or unidentified toxic agent.Without being bound by any one specific theory for mechanism of action,exosomes are shed from damaged cells and in the process compartmentalizespecific contents of the cell including both membrane components andengulfed cytoplasmic contents. Cells exposed to toxic agents/chemicalsmay increase exosome shedding to expel toxic agents or metabolitesthereof, thus resulting in increased exosome levels. Thus, monitoring anexosome and/or bio-signature allows assessment of an individual'sresponse to potential toxic agent(s).

Furthermore, an exosome can be used to identify states of drug-inducedtoxicity or the organ injured, by detecting one or more specificantigen, binding agent, biomarker, or any combination thereof of theexosome. Therefore, the exosome, or exosome bio-signature can be used tomonitor an individual for acute, chronic, or occupational exposures toany number of toxic agents including, but not limited to, drugs,antibiotics, industrial chemicals, toxic antibiotic metabolites, herbs,household chemicals, and chemicals produced by other organisms, eithernaturally occurring or synthetic in nature.

In addition, an exosome bio-signature can be used to identify conditionsor diseases, including cancers of unknown origin, also known as cancersof unknown primary (CUP). For example, an exosome may be isolated from abiological sample as previously described to arrive at a heterogeneouspopulation of exosomes. The heterogeneous population of exosomes canthen be applied to surfaces coated with specific binding agents designedto rule out or identify antigen specific characteristics of the exosomepopulation that are specific to a given cell-of-origin. Further, asdescribed above, the bio-signature of a specific cell-of-origin exosomecan correlate with the cancerous state of cells. Compounds that inhibitcancer in a subject may cause a change, e.g., a change in bio-signatureof specific cell-of-origin exosome, which can be monitored by serialisolation of a cell-of-origin exosome over time and treatment course.

Alternatively, an exosome bio-signature can be used to assess theefficacy of a therapy, e.g., chemotherapy, radiation therapy, surgery,or any other therapeutic approach useful for inhibiting cancer in asubject. In addition, an exosome bio-signature can be used in ascreening assay to identify candidate or test compounds or agents (e.g.,proteins, peptides, peptidomimetics, peptoids, small molecules or otherdrugs) that have a modulatory effect on the bio-signature of a specificcell-of-origin exosome. Compounds identified via such screening assaysmay be useful, for example, for modulating, e.g., inhibiting,ameliorating, treating, or preventing conditions or diseases.

For example, a bio-signature for an exosome can be obtained from apatient who is undergoing successful treatment for a particular cancer.Cells from a cancer patient not being treated with the same drug can becultured and exosomes from the cultures obtained for determiningbio-signatures. The cells can be treated with test compounds and thebio-signature of the exosomes from the cultures can be compared to thebio-signature of the exosomes obtained from the patient undergoingsuccessful treatment. The test compounds that results in exosomebio-signatures that are similar to those of the patient undergoingsuccessful treatment can be selected for further studies.

The bio-signature of a specific cell-of-origin exosome can also be usedto monitor the influence of an agent (e.g., drug compounds) on thebio-signature in clinical trials. Monitoring an exosome bio-signaturecan also be used in a method of assessing the efficacy of a testcompound, such as a test compound for inhibiting cancer cells.

An exosome bio-signature can also be used to determine the effectivenessof a particular therapeutic intervention (pharmaceutical ornon-pharmaceutical) and to alter the intervention to 1) reduce the riskof developing adverse outcomes, 2) enhance the effectiveness of theintervention or 3) identify resistant states. Thus, in addition todiagnosing or confirming the presence of or risk for developing adisease, condition or a syndrome, the methods and compositions disclosedherein also provide a system for optimizing the treatment of a subjecthaving such a disease, condition or syndrome. For example, atherapy-related approach to treating a disease, condition or syndrome byintegrating diagnostics and therapeutics to improve the real-timetreatment of a subject can be determined by identifying thebio-signature of an exosome.

Tests that identify an exosome bio-signature can be used to identifywhich patients are most suited to a particular therapy, and providefeedback on how well a drug is working, so as to optimize treatmentregimens. For example, in pregnancy-induced hypertension and associatedconditions, therapy-related diagnostics can flexibly monitor changes inimportant parameters (e.g., cytokine and/or growth factor levels) overtime, to optimize treatment.

Within the clinical trial setting of investigational agents as definedby the FDA, MDA, EMA, USDA, and EMEA, therapy-related diagnostics asdetermined by a bio-signature disclosed herein, can provide keyinformation to optimize trial design, monitor efficacy, and enhance drugsafety. For instance, for trial design, therapy-related diagnostics canbe used for patient stratification, determination of patient eligibility(inclusion/exclusion), creation of homogeneous treatment groups, andselection of patient samples that are optimized to a matched casecontrol cohort. Such therapy-related diagnostic can therefore providethe means for patient efficacy enrichment, thereby minimizing the numberof individuals needed for trial recruitment. For example, for efficacy,therapy-related diagnostics are useful for monitoring therapy andassessing efficacy criteria. Alternatively, for safety, therapy-relateddiagnostics can be used to prevent adverse drug reactions or avoidmedication error and monitor compliance with the therapeutic regimen.

Therefore, an exosomal bio-signature can be used to monitor drugefficacy, determine response or resistance to a given drug, and therebyenhance drug safety. For example, in colon cancer, exosomes aretypically shed from colon cancer cells and can be isolated from theperipheral blood and used to isolate one or more biomarkers (e.g., KRASmRNA). In the case of mRNA biomarkers, the mRNA can be reversetranscribed into cDNA and sequenced (e.g., by Sanger sequencing) todetermine if there are mutations present that confer resistance to adrug (e.g., cetuximab or panitumimab).

In another example, exosomes that are specifically shed from lung cancercells are isolated from a biological sample and used to isolate a lungcancer biomarker, e.g., EGFR mRNA. The EGFR mRNA is processed to cDNAand sequenced to determine if there are EGFR mutations present that showresistance or response to specific drugs or treatments for lung cancer.

One or more exosome bio-signatures can be grouped so that informationobtained about the set of bio-signatures in a particular group providesa reasonable basis for making a clinically relevant decision, such asbut not limited to a diagnosis, prognosis, or management of treatment,such as treatment selection.

As with most diagnostic markers, it is often desirable to use the fewestnumber of markers sufficient to make a correct medical judgment. Thisprevents a delay in treatment pending further analysis as wellinappropriate use of time and resources.

Also disclosed herein are methods of conducting retrospective analysison samples (e.g., serum and tissue biobanks) for the purpose ofcorrelating qualitative and quantitative properties, such as exosomebio-signatures, with clinical outcomes in terms of disease state,disease stage, progression, prognosis; therapeutic efficacy orselection; or physiological conditions. Furthermore, methods andcompositions disclosed herein are utilized for conducting prospectiveanalysis on a sample (e.g., serum and/or tissue collected fromindividuals in a clinical trial) for the purpose of correlatingqualitative and quantitative exosome bio-signatures with clinicaloutcomes in terms of disease state, disease stage, progression,prognosis; therapeutic efficacy or selection; or physiologicalconditions can also be performed. As used herein, exosome bio-signaturescan be to cell-of-origin specific exosomes. Furthermore, bio-signaturescan be determined based on an exosome surface marker profile and/orexosome contents (e.g., biomarkers).

An exosome bio-signature can comprise at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75, or 100characteristics. A bio-signature with more than one exosomalcharacteristic, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75, or 100 characteristics,may provide higher sensitivity, specificity, or both, in determining aphenotype. For example, assessing a plurality of exosomalcharacteristics can provide increased sensitivity, specificity, or both,as compared to assessing less than a plurality of exosomalcharacteristics.

A bio-signature comprising more than one exosomal characteristic can beused to characterize a phenotype with at least 60, 61, 62, 63, 64, 65,66, 67, 68, 69, or 70% sensitivity, such as with at least 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, or 87% sensitivity.For example, the phenotype can be characterized with at least 87.1,87.2, 87.3, 87.4, 87.5, 87.6, 87.7, 87.8, 87.9, 88.0, or 89%sensitivity, such as at least 90% sensitivity. The phenotype can becharacterized with at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%sensitivity.

The bio-signature can be used to characterize a phenotype of a subjectwith at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97% specificity,such as with at least 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8,97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9,99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%specificity.

The phenotype can also be characterized using a bio-signature with atleast 70% sensitivity and at least 80, 90, 95, 99, or 100% specificity;at least 75% sensitivity and at least 80, 90, 95, 99, or 100%specificity; at least 80% sensitivity and at least 80, 85, 90, 95, 99,or 100% specificity; at least 85% sensitivity and at least 80, 85, 90,95, 99, or 100% specificity; at least 86% sensitivity and at least 80,85, 90, 95, 99, or 100% specificity; at least 87% sensitivity and atleast 80, 85, 90, 95, 99, or 100% specificity; at least 88% sensitivityand at least 80, 85, 90, 95, 99, or 100% specificity; at least 89%sensitivity and at least 80, 85, 90, 95, 99, or 100% specificity; atleast 90% sensitivity and at least 80, 85, 90, 95, 99, or 100%specificity; at least 95% sensitivity and at least 80, 85, 90, 95, 99,or 100% specificity; at least 99% sensitivity and at least 80, 85, 90,95, 99, or 100% specificity; or at least 100% sensitivity and at least80, 85, 90, 95, 99, or 100% specificity.

Furthermore, the confidence level for determining the specificity,sensitivity, or both, may be with at least 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% confidence.

Bio-Signatures: Exosomal Biomarkers

An exosome bio-signature can comprise one or more biomarkers. Anexosomal biomarker can be any component present in an exosome or on theexosome, such as any nucleic acid (e.g. RNA or DNA); protein, peptide,polypeptide, antigen, lipid, carbohydrate, or proteoglycan.

The bio-signature can include the presence or absence, expression level,mutational state, genetic variant state, or any modification (such asepigentic modification, post-translation modification) of a biomarker(e.g. any one or more biomarker listed in FIGS. 1, 3-60). The expressionlevel of a biomarker can be compared to a control or reference, todetermine the overexpression or underexpression (or upregulation ordownregulation) of a biomarker in a sample. The control or referencelevel can be the amount of a biomarker, such as a miRNA in a controlsample, such as a sample from a subject that does not have or exhibitthe condition or disease, and further described below.

The nucleic acid can be any RNA or DNA species. For example, thebiomarker can be mRNA, miRNA, small nucleolar RNAs (snoRNA), smallnuclear RNAs (snRNA), ribosomal RNAs (rRNA), heterogeneous nuclear RNA(hnRNA), ribosomal RNAS (rRNA), siRNA, transfer RNAs (tRNA), or shRNA.The DNA can be double-stranded DNA, single stranded DNA, complementaryDNA, or noncoding DNA.

In addition, the biomarker can be a polypeptide, peptides or protein,such as the modification state, truncations, mutations, expression level(such as overexpression or underexpression as compared to a referencelevel), and post-translational modifications, such as described above.

An exosome bio-signature may include a number of the same type ofbiomarkers (e.g., two different mRNAs, each corresponding to a differentgene) or one or more of different types of biomarkers (e.g. mRNAs,miRNAs, proteins, peptides, ligands, and antigens).

One or more exosome bio-signatures can comprise at least one biomarkerselected from those listed in FIGS. 1, 3-60. A specific cell-of-originbio-signature may include one or more biomarkers. FIGS. 3-58 depicttables which lists a number of disease or condition specific biomarkersthat can be derived and analyzed from an exosome. The biomarker can alsobeCD24, midkine, hepcidin, TMPRSS2-ERG, PCA-3, PSA, EGFR, EGFRvIII, BRAFvariant, MET, cKit, PDGFR, Wnt, beta-catenin, K-ras, H-ras, N-ras, Raf,N-myc, c-myc, IGFR, PI3K, Akt, BRCA1, BRCA2, PTEN, VEGFR-2, VEGFR-1,Tie-2, TEM-1, CD276, HER-2, HER-3, or HER-4. The biomarker can also beannexin V, CD63, Rab-5b, or caveolin, or a miRNA, such as let-7a;miR-15b; miR-16; miR-19b; miR-21; miR-26a; miR-27a; miR-92; miR-93;miR-320 or miR-20. The biomarker can also be of any gene or fragmentthereof as disclosed in PCT Publication No. WO2009/100029, such as thoselisted in Tables 3-15.

Other biomarkers useful for assessment in methods and compositionsdisclosed herein can include those associated with conditions orphysiological states as disclosed in Rajendran et al., Proc Natl AcadSci USA 2006; 103:11172-11177, Taylor et al., Gynecol Oncol 2008;110:13-21, Zhou et al., Kidney Int 2008; 74:613-621, Buning et al.,Immunology 2008, Prado et al. J Immunol 2008; 181:1519-1525, Vella etal. (2008) Vet Immunol Immunopathol 124(3-4): 385-93, Gould et al.(2003). Proc Natl Acad Sci USA 100(19): 10592-7, Fang et al. (2007).PLoS Biol 5(6): e158, Chen, B. J. and R. A. Lamb (2008). Virology372(2): 221-32, Bhatnagar, S, and J. S. Schorey (2007). J Biol Chem282(35): 25779-89, Bhatnagar et al. (2007) Blood 110(9): 3234-44,Yuyama, et al. (2008). J Neurochem 105(1): 217-24, Gomes et al. (2007).Neurosci Lett. 428(1): 43-6, Nagahama et al. (2003). Autoimmunity 36(3):125-31, Taylor, D. D., S. Akyol, et al. (2006). J Immunol 176(3):1534-42, Peche, et al. (2006). Am J Transplant 6(7): 1541-50, lero, M.,M. Valenti, et al. (2008). Cell Death and Differentiation 15: 80-88,Gesierich, S., I. Berezoversuskiy, et al. (2006), Cancer Res 66(14):7083-94, Clayton, A., A. Turkes, et al. (2004). Faseb J 18(9): 977-9,Skriner., K. Adolph, et al. (2006). Arthritis Rheum 54(12): 3809-14,Brouwer, R., G. J. Pruijn, et al. (2001). Arthritis Res 3(2): 102-6,Kim, S. H., N. Bianco, et al. (2006). Mol Ther 13(2): 289-300, Evans, C.H., S. C. Ghivizzani, et al. (2000). Clin Orthop Relat Res (379 Suppl):S300-7, Zhang, H. G., C. Liu, et al. (2006). J Immunol 176(12): 7385-93,Van Niel, G., J. Mallegol, et al. (2004). Gut 52: 1690-1697, Fiasse, R.and O. Dewit (2007). Expert Opinion on Therapeutic Patents 17(12):1423-1441(19).

A biomarker that can be derived and analyzed from an exosome includes,but is not limited to, the presence or absence, expression level,mutations (for example genetic mutations, such as deletions,translocations, duplications, nucleotide or amino acid substitutions,and the like) of miRNA (miR) and miRNA*nonsense (miR*), and other RNAs(including, but not limited to, mRNA, preRNA, preRNA, hnRNA, snRNA,siRNA, shRNA), DNA, proteins, peptides, and ligands. Any epigeneticmodulation or copy number variation of a biomarker can also be analyzed.A miRNA biomarker includes not only its miRNA and microRNA*nonsense, butits precursor molecules: pri-microRNAs (pri-miRs) and pre-microRNAs(pre-miRs) are also included as biomarkers. The sequence of a miRNA canbe obtained from publicly available databases such ashttp://www.mirbase.org/, http://www.microrna.org/, or any othersavailable.

The one or more biomarkers analyzed from an exosome can be indicative ofa particular tissue or cell of origin, disease, or physiological state,as further described below. Furthermore, the presence, absence orexpression level of one or more of the biomarkers described herein canbe correlated to a phenotype of a subject, including a disease,condition, prognosis or drug efficacy. The specific biomarker andbio-signature set forth below constitute non-inclusive examples for eachof the diseases, condition comparisons, conditions, and/or physiologicalstates. Furthermore, the one or more biomarker assessed for a phenotypecan be a cell-of-origin specific exosome, such as those described above.

The one or more miRNAs used to characterize a phenotype may be selectedfrom those disclosed in PCT Publication No. WO2009/036236. For example,one or more miRNAs listed in Tables I-VI (FIGS. 6-11) can be used tocharacterize colon adenocarcinoma, colorectal cancer, prostate cancer,lung cancer, breast cancer, b-cell lymphoma, pancreatic cancer, diffuselarge BCL cancer, CLL, bladder cancer, renal cancer, hypoxia-tumor,uterine leiomyomas, ovarian cancer, hepatitis C virus-associatedhepatocellular carcinoma, ALL, Alzheimer's disease, myelofibrosis,myelofibrosis, polycythemia vera, thrombocythemia, HIV, or HIV-Ilatency, as further described herein.

The one or more miRNAs can be detected in plasma exosomes. The one ormore miRNAs can be miR-223, miR-484, miR-191, miR-146a, miR-016,miR-026a, miR-222, miR-024, miR-126, and miR-32. One or more miRNAs canalso be detected in PBMC. The one or more miRNAs can be miR-223,miR-150, miR-146b, miR-016, miR-484, miR-146a, miR-191, miR-026a,miR-019b, or miR-020a. The one or more miRNAs can be used tocharacterize a particular disease or condition. For example, for thedisease bladder cancer, one or more miRNAs can be detected, such asmiR-223, miR-26b, miR-221, miR-103-1, miR-185, miR-23b, miR-203,miR-17-5p, miR-23a, miR-205 or any combination thereof. The one or moremiRNAs may be upregulated or overexpressed.

In some embodiments, the one or more miRNAs is used to characterizehypoxia-tumor. The one or more miRNA may be miR-23, miR-24, miR-26,miR-27, miR-103, miR-107, miR-181, miR-210, or miR-213, and may beupregulated. One or more miRNAs can also be used to characterize uterineleiomyomas. For example, the one or more miRNAs used to characterize auterine leiomyoma may be a let-7 family member, miR-21, miR-23b,miR-29b, or miR-197. The miRNA can be upregulated.

Myelofibrosis can also be characterized by one or more miRNAs, such asmiR-190, which can be upregulated; miR-31, miR-150 and miR-95, which canbe downregulated, or any combination thereof. Furthermore,myelofibrosis, polycythemia vera or thrombocythemia can also becharacterized by detecting one or more miRNAs, such as, but not limitedto, miR-34a, miR-342, miR-326, miR-105, miR-149, miR-147, or anycombination thereof. The one or more miRNAs may be down-regulated.

Other examples of phenotypes that can be characterized by assessing anexosome for one or more biomarkers are further described herein.

The one or more biomarkers can be detected by a probe. A probe cancomprise of an oligonucleotide, such as DNA or RNA, an aptamer,monoclonal antibody, polyclonal antibody, Fabs, Fab′, single chainantibody, synthetic antibody, peptoid, zDNA, peptide nucleic acid (PNA),locked nucleic acid (LNA), lectin, synthetic or naturally occurringchemical compound (including but not limited to a drug or labelingreagent), dendrimer, or any combination thereof. The probe can bedirectly detected, for example by being directly labeled, or beindirectly detected, such as through a labeing reagent. The probe canselectively hybridize to a biomarker. For example, a probe that is anoligonucleotide can selectively hybridize to a miRNA biomarker.

Breast Cancer

Breast cancer specific biomarkers can include one or more (for example,2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs, underexpressed miRs,mRNA, genetic mutations, proteins, ligands, peptides, snoRNA, or anycombination thereof, such as listed in FIG. 3.

One or more breast cancer specific biomarker can be assessed to providea breast cancer specific exosome bio-signature. For example, thebio-signature can comprise one or more overexpressed miRs, including butnot limited to, miR-21, miR-155, miR-206, miR-122a, miR-210, miR-21,miR-21, miR-155, miR-206, miR-122a, miR-210, or miR-21, or anycombination thereof.

The bio-signature can also comprise one or more underexpressed miRs suchas, but not limited to, let-7, miR-10b, miR-125a, miR-125b, miR-145,miR-143, miR-145, miR-16, let-7, let-7, let-7, miR-10b, miR-125a,miR-125b, or miR-145, or any combination thereof.

The mRNAs that may be analyzed can include, but are not limited to, ER,PR, HER2, MUC1, or EGFR, or any combination thereof. Mutationsincluding, but not limited to, those related to KRAS, B-Raf, or CYP2D6,or any combination thereof can also be used as specific biomarkers fromexosomes for breast cancer. In addition, a protein, ligand, or peptidethat can be used as biomarkers from exosomes that are specific to breastcancer includes, but are not limited to, hsp70, MART-1, TRP, HER2,hsp70, MART-1, TRP, HER2, ER, PR, Class III b-tubulin, or VEGFA, or anycombination thereof. Furthermore the snoRNA that can be used as anexosomal biomarker for breast cancer include, but are not limited to,GAS5. The gene fusion ETV6-NTRK3 can also be used a biomarker for breastcancer.

Also provided herein is an isolated exosome comprising one or morebreast cancer specific biomarkers, such as ETV6-NTRK3, or biomarkerslisted in FIG. 3 and in FIG. 1 for breast cancer. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more breast cancer specific biomarkers, such asETV6-NTRK3, or biomarkers listed in FIG. 3 and in FIG. 1 for breastcancer. The composition can comprise a substantially enriched populationof exosomes, wherein the population of exosomes is substantiallyhomogeneous for breast cancer specific exosomes or exosomes comprisingone or more breast cancer specific biomarkers, such as ETV6-NTRK3, orbiomarkers listed in FIG. 3 and in FIG. 1 for breast cancer.

One or more breast cancer specific biomarkers, such as ETV6-NTRK3, orbiomarkers listed in FIG. 3 and in FIG. 1 for breast cancer can also bedetected by one or more systems disclosed herein, for characterizing abreast cancer. For example, a detection system can comprise one or moreprobes to detect one or more breast cancer specific biomarkers, such asETV6-NTRK3, or biomarkers listed in FIG. 3 and in FIG. 1 for breastcancer, of one or more exosomes of a biological sample.

Ovarian Cancer

Ovarian cancer specific biomarkers from exosomes can include one or more(for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 4,and can be used to create a ovarian cancer specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-200a, miR-141,miR-200c, miR-200b, miR-21, miR-141, miR-200a, miR-200b, miR-200c,miR-203, miR-205, miR-214, miR-199″, or miR-215, or any combinationthereof. The bio-signature can also comprise one or more underexpressedmiRs such as, but not limited to, miR-199a, miR-140, miR-145, miR-100,miR-let-7 cluster, or miR-125b-1, or any combination thereof. The one ormore mRNAs that may be analyzed can include, but are not limited to,ERCC1, ER, TOPO1, TOP2A, AR, PTEN, HER2/neu, CD24 or EGFR, or anycombination thereof.

A biomarker mutation for ovarian cancer that can be assessed in anexosome includes, but is not limited to, a mutation of KRAS, mutation ofB-Raf, or any combination of mutations specific for ovarian cancer. Theprotein, ligand, or peptide that can be assessed in an exosome caninclude, but is not limited to, VEGFA, VEGFR2, or HER2, or anycombination thereof. Furthermore, an exosome isolated or assayed can beovarian cancer cell specific, or derived from ovarian cancer cells.

Also provided herein is an isolated exosome comprising one or moreovarian cancer specific biomarkers, such as CD24, those listed in FIG. 4and in FIG. 1 for ovarian cancer. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or moreovarian cancer specific biomarkers, such as CD24, those listed in FIG. 4and in FIG. 1 for ovarian cancer. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for ovarian cancer specificexosomes or exosomes comprising one or more ovarian cancer specificbiomarkers, such as CD24, those listed in FIG. 4 and in FIG. 1 forovarian cancer.

One or more ovarian cancer specific biomarkers, such as CD24, thoselisted in FIG. 4 and in FIG. 1 for ovarian cancer can also be detectedby one or more systems disclosed herein, for characterizing an ovariancancer. For example, a detection system can comprise one or more probesto detect one or more ovarian cancer specific biomarkers, such as CD24,those listed in FIG. 4 and in FIG. 1 for ovarian cancer, of one or moreexosomes of a biological sample.

Lung Cancer

Lung cancer specific biomarkers from exosomes can include one or more(for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 5,and can be used to create a lung cancer specific exosome bio-signature.

The bio-signature can comprise one or more overexpressed miRs, such as,but not limited to, miR-21, miR-205, miR-221 (protective), let-7a(protective), miR-137 (risky), miR-372 (risky), or miR-122a (risky), orany combination thereof. The bio-signature can comprise one or moreupregulated or overexpressed miRNAs, such as miR-17-92, miR-19a, miR-21,miR-92, miR-155, miR-191, miR-205 or miR-210; one or more downregulatedor underexpressed miRNAs, such as miR-let-7, or any combination thereof.

The one or more mRNAs that may be analyzed can include, but are notlimited to, EGFR, PTEN, RRM1, RRM2, ABCB1, ABCG2, LRP, VEGFR2, VEGFR3,class III b-tubulin, or any combination thereof.

A biomarker mutation for lung cancer that can be assessed in an exosomeincludes, but is not limited to, a mutation of EGFR, KRAS, B-Raf,UGT1A1, or any combination of mutations specific for lung cancer. Theprotein, ligand, or peptide that can be assessed in an exosome caninclude, but is not limited to, KRAS, hENT1, or any combination thereof.

The biomarker can also be midkine (MK or MDK). Furthermore, an exosomeisolated or assayed can be lung cancer cell specific, or derived fromlung cancer cells.

Also provided herein is an isolated exosome comprising one or more lungcancer specific biomarkers, such as RLF-MYCL1, TGF-ALK, or CD74-ROS1, orthose listed in FIG. 5 and in FIG. 1 for lung cancer. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more lung cancer specific biomarkers, such asRLF-MYCL1, TGF-ALK, or CD74-ROS1, or those listed in FIG. 5 and in FIG.1 for lung cancer. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for lung cancer specific exosomes or exosomescomprising one or more lung cancer specific biomarkers, such asRLF-MYCL1, TGF-ALK, or CD74-ROS1, or those listed in FIG. 5 and in FIG.1 for lung cancer.

One or more lung cancer specific biomarkers, such as RLF-MYCL1, TGF-ALK,or CD74-ROS1, or those listed in FIG. 5 and in FIG. 1 for lung cancercan also be detected by one or more systems disclosed herein, forcharacterizing a lung cancer. For example, a detection system cancomprise one or more probes to detect one or more lung cancer specificbiomarkers, such as RLF-MYCL1, TGF-ALK, or CD74-ROS1, or those listed inFIG. 5 and in FIG. 1 for lung cancer, of one or more exosomes of abiological sample.

Colon Cancer

Colon cancer specific biomarkers from exosomes can include one or more(for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 6,and can be used to create a colon cancer specific exosome bio-signature.For example, the bio-signature can comprise one or more overexpressedmiRs, such as, but not limited to, miR-24-1, miR-29b-2, miR-20a,miR-10a, miR-32, miR-203, miR-106a, miR-17-5p, miR-30c, miR-223,miR-126, miR-128b, miR-21, miR-24-2, miR-99b, miR-155, miR-213, miR-150,miR-107, miR-191, miR-221, miR-20a, miR-510, miR-92, miR-513, miR-19a,miR-21, miR-20, miR-183, miR-96, miR-135b, miR-31, miR-21, miR-92,miR-222, miR-181b, miR-210, miR-20a, miR-106a, miR-93, miR-335, miR-338,miR-133b, miR-346, miR-106b, miR-153a, miR-219, miR-34a, miR-99b,miR-185, miR-223, miR-211, miR-135a, miR-127, miR-203, miR-212, miR-95,or miR-17-5p, or any combination thereof. The bio-signature can alsocomprise one or more underexpressed miRs such as miR-143, miR-145,miR-143, miR-126, miR-34b, miR-34c, let-7, miR-9-3, miR-34a, miR-145,miR-455, miR-484, miR-101, miR-145, miR-133b, miR-129, miR-124a,miR-30-3p, miR-328, miR-106a, miR-17-5p, miR-342, miR-192, miR-1,miR-34b, miR-215, miR-192, miR-301, miR-324-5p, miR-30a-3p, miR-34c,miR-331, or miR-148b, or any combination thereof.

The one or more biomarker can be an upregulated or overexpressed miRNA,such as miR-20a, miR-21, miR-106a, miR-181b or miR-203, forcharacterizing a colon adenocarcinoma. The one or more biomarker can beused to characterize a colorectal cancer, such as an upregulated oroverexpressed miRNA selected from the group consisting of: miR-19a,miR-21, miR-127, miR-31, miR-96, miR-135b and miR-183, a downregulatedor underexpressed miRNA, such as miR-30c, miR-133a, mir143, miR-133b ormiR-145, or any combination thereof.

The one or more mRNAs that may be analyzed can include, but are notlimited to, EFNB1, ERCC1, HER2, VEGF, or EGFR, or any combinationthereof. A biomarker mutation for colon cancer that can be assessed inan exosome includes, but is not limited to, a mutation of EGFR, KRAS,VEGFA, B-Raf, APC, or p53, or any combination of mutations specific forcolon cancer. The protein, ligand, or peptide that can be assessed in anexosome can include, but is not limited to, AFRs, Rabs, ADAM10, CD44,NG2, ephrin-B1, MIF, b-catenin, Junction, plakoglobin, glalectin-4,RACK1, tetrspanin-8, FasL, TRAIL, A33, CEA, EGFR, dipeptidase 1, hsc-70,tetraspanins, ESCRT, TS, PTEN, or TOPO1, or any combination thereof.Furthermore, an exosome isolated or assayed can be colon cancer cellspecific, or derived from colon cancer cells.

Also provided herein is an isolated exosome comprising one or more coloncancer specific biomarkers, such as listed in FIG. 6 and in FIG. 1 forcolon cancer. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more colon cancer specificbiomarkers, such as listed in FIG. 6 and in FIG. 1 for colon cancer. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for colon cancer specific exosomes or exosomes comprisingone or more colon cancer specific biomarkers, such as listed in FIG. 6and in FIG. 1 for colon cancer.

One or more colon cancer specific biomarkers, such as listed in FIG. 6and in FIG. 1 for colon cancer can also be detected by one or moresystems disclosed herein, for characterizing a colon cancer. Forexample, a detection system can comprise one or more probes to detectone or more colon cancer specific biomarkers, such as listed in FIG. 6and in FIG. 1 for colon cancer, of one or more exosomes of a biologicalsample.

Adenoma Versus Hyperplastic Polyp

Adenoma versus hyperplastic polyp specific biomarkers from exosomes caninclude one or more (for example, 2, 3, 4, 5, 6, 7, 8, or more)overexpressed miRs, underexpressed miRs, mRNAs, genetic mutations,proteins, ligands, peptides, or any combination thereof, such as listedin FIG. 7, and can be used to create an adenoma versus hyperplasticpolyp specific exosome bio-signature. For example, the one or more mRNAsthat may be analyzed can include, but are not limited to, ABCA8,KIAA1199, GCG, MAMDC2, C2orf32, 229670_at, IGF1, PCDH7, PRDX6, PCNA,COX2, or MUC6, or any combination thereof.

A biomarker mutation to distinguish for adenoma versus hyperplasticpolyp that can be assessed in an exosome includes, but is not limitedto, a mutation of KRAS, mutation of B-Raf, or any combination ofmutations specific for distinguishing between adenoma versushyperplastic polyp. The protein, ligand, or peptide that can be assessedin an exosome can include, but is not limited to, hTERT.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between an adenoma and ahyperplastic polyp, such as listed in FIG. 7. A composition comprisingthe isolated exosome is also provided. Accordingly, in some embodiments,the composition comprises a population of exosomes comprising one ormore specific biomarkers for distinguishing between an adenoma and ahyperplastic polyp, such as listed in FIG. 7. The composition cancomprise a substantially enriched population of exosomes, wherein thepopulation of exosomes is substantially homogeneous for having one ormore specific biomarkers for distinguishing between an adenoma and ahyperplastic polyp, such as listed in FIG. 7.

One or more specific biomarkers for distinguishing between an adenomaand a hyperplastic polyp, such as listed in FIG. 7 can also be detectedby one or more systems disclosed herein, for distinguishing between anadenoma and a hyperplastic polyp. For example, a detection system cancomprise one or more probes to detect one or more specific biomarkersfor distinguishing between an adenoma and a hyperplastic polyp, such aslisted in FIG. 7, of one or more exosomes of a biological sample.

Irritable Bowel Disease (IBD)

IBD versus normal biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 8,and can be used to create a IBD versus normal specific exosomebio-signature. For example, the one or more mRNAs that may be analyzedcan include, but are not limited to, REG1A, MMP3, or any combinationthereof.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between IBD and a normal sample,such as listed in FIG. 8. A composition comprising the isolated exosomeis also provided. Accordingly, in some embodiments, the compositioncomprises a population of exosomes comprising one or more specificbiomarkers for distinguishing between IBD and a normal sample, such aslisted in FIG. 8. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for having one or more specific biomarkers fordistinguishing between IBD and a normal sample, such as listed in FIG.8.

One or more specific biomarkers for distinguishing between IBD and anormal sample, such as listed in FIG. 8 can also be detected by one ormore systems disclosed herein, for distinguishing between IBD and anormal sample. For example, a detection system can comprise one or moreprobes to detect one or more specific biomarkers for distinguishingbetween IBD and a normal sample, such as listed in FIG. 8, of one ormore exosomes of a biological sample.

Adenoma Versus Colorectal Cancer (CRC)

Adenoma versus CRC specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 9,and can be used to create a Adenoma versus CRC specific exosomebio-signature. For example, the one or more mRNAs that may be analyzedcan include, but are not limited to, GREM1, DDR2, GUCY1A3, TNS1,ADAMTS1, FBLN1, FLJ38028, RDX, FAM129A, ASPN, FRMD6, MCC, RBMS1, SNA12,MEIS1, DOCK10, PLEKHC1, FAM126A, TBC1D9, VWF, DCN, ROBO1, MSRB3, LATS2,MEF2C, IGFBP3, GNB4, RCN3, AKAP12, RFTN1, 226834_at, COL5A1, GNG2,NR3C1*, SPARCL1, MAB21L2, AXIN2, 236894_at, AEBP1, AP1S2, C10orf56,LPHN2, AKT3, FRMD6, COL15A1, CRYAB, COL14A1, LOC286167, QKI, WWTR1,GNG11, PAPPA, or ELDT1, or any combination thereof.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between an adenoma and a CRC,such as listed in FIG. 9. A composition comprising the isolated exosomeis also provided. Accordingly, in some embodiments, the compositioncomprises a population of exosomes comprising one or more specificbiomarkers for distinguishing between an adenoma and a CRC, such aslisted in FIG. 9. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for having one or more specific biomarkers fordistinguishing between an adenoma and a CRC, such as listed in FIG. 9.

One or more specific biomarkers for distinguishing between an adenomaand a CRC, such as listed in FIG. 9 can also be detected by one or moresystems disclosed herein, for distinguishing between an adenoma and aCRC. For example, a detection system can comprise one or more probes todetect one or more specific biomarkers for distinguishing between anadenoma and a CRC, such as listed in FIG. 9, of one or more exosomes ofa biological sample.

IBD Versus CRC

IBD versus CRC specific biomarkers from exosomes can include one or more(for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 10,and can be used to create a IBD versus CRC specific exosomebio-signature. For example, the one or more mRNAs that may be analyzedcan include, but are not limited to, 227458_at, INDO, CXCL9, CCR2, CD38,RARRES3, CXCL10, FAM26F, TNIP3, NOS2A, CCRL1, TLR8, IL18BP, FCRL5,SAMD9L, ECGF1, TNFSF13B, GBPS, or GBP1, or any combination thereof.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between IBD and a CRC, such aslisted in FIG. 10. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more specific biomarkers fordistinguishing between IBD and a CRC, such as listed in FIG. 10. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for having one or more specific biomarkers fordistinguishing between IBD and a CRC, such as listed in FIG. 10.

One or more specific biomarkers for distinguishing between IBD and aCRC, such as listed in FIG. 10 can also be detected by one or moresystems disclosed herein, for distinguishing between IBD and a CRC. Forexample, a detection system can comprise one or more probes to detectone or more specific biomarkers for distinguishing between IBD and aCRC, such as listed in FIG. 10, of one or more exosomes of a biologicalsample.

CRC Dukes B Versus Dukes C-D

CRC Dukes B versus Dukes C-D specific biomarkers from exosomes caninclude one or more (for example, 2, 3, 4, 5, 6, 7, 8, or more)overexpressed miRs, underexpressed miRs, mRNAs, genetic mutations,proteins, ligands, peptides, snoRNA, or any combination thereof, such aslisted in FIG. 11, and can be used to create a CRC D-B versus C-Dspecific exosome bio-signature. For example, the one or more mRNAs thatmay be analyzed can include, but are not limited to, TMEM37*, IL33, CA4,CCDC58, CLIC6, VERSUSNL1, ESPN, APCDD1, C13orf18, CYP4X1, ATP2A3,LOC646627, MUPCDH, ANPEP, C1orf115, HSD3B2, GBA3, GABRB2, GYLTL1B, LYZ,SPC25, CDKN2B, FAM89A, MOGAT2, SEMA6D, 229376_at, TSPAN5, IL6R, orSLC26A2, or any combination thereof.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between CRC Dukes B and a CRCDukes C-D, such as listed in FIG. 11. A composition comprising theisolated exosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morespecific biomarkers for distinguishing between CRC Dukes B and a CRCDukes C-D, such as listed in FIG. 11. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for having one or more specificbiomarkers for distinguishing between CRC Dukes B and a CRC Dukes C-D,such as listed in FIG. 11.

One or more specific biomarkers for distinguishing between CRC Dukes Band a CRC Dukes C-D, such as listed in FIG. 11 can also be detected byone or more systems disclosed herein, for distinguishing between CRCDukes B and a CRC Dukes C-D. For example, a detection system cancomprise one or more probes to detect one or more specific biomarkersfor distinguishing between CRC Dukes B and a CRC Dukes C-D, such aslisted in FIG. 11, of one or more exosomes of a biological sample.

Adenoma with Low Grade Dysplasia Versus Adenoma with High GradeDysplasia

Adenoma with low grade dysplasia versus adenoma with high gradedysplasia specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 12,and can be used to create an adenoma low grade dysplasia versus adenomahigh grade dysplasia specific exosome bio-signature. For example, theone or mRNAs that may be analyzed can include, but are not limited to,SI, DMBT1, CFI*, AQP1, APOD, TNFRSF17, CXCL10, CTSE, IGHA1, SLC9A3,SLC7A1, BATF2, SOCS1, DOCK2, NOS2A, HK2, CXCL2, IL15RA, POU2AF1, CLEC3B,ANI3BP, MGC13057, LCK*, C4BPA, HOXC6, GOLT1A, C2orf32, IL10RA,240856_at, SOCS3, MEIS3P1, HIPK1, GLS, CPLX1, 236045_x_at, GALC, AMN,CCDC69, CCL28, CPA3, TRIB2, HMGA2, PLCL2, NR3C1, EIF5A, LARP4,RP5-1022P6.2, PHLDB2, FKBP1B, INDO, CLDN8, CNTN3, PBEF1, SLC16A9,CDC25B, TPSB2, PBEF1, ID4, GJB5, CHN2, LIMCH1, or CXCL9, or anycombination thereof.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between adenoma with low gradedysplasia and adenoma with high grade dysplasia, such as listed in FIG.12. A composition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more specific biomarkers fordistinguishing between adenoma with low grade dysplasia and adenoma withhigh grade dysplasia, such as listed in FIG. 12. The composition cancomprise a substantially enriched population of exosomes, wherein thepopulation of exosomes is substantially homogeneous for having one ormore specific biomarkers for distinguishing between adenoma with lowgrade dysplasia and adenoma with high grade dysplasia, such as listed inFIG. 12.

One or more specific biomarkers for distinguishing between adenoma withlow grade dysplasia and adenoma with high grade dysplasia, such aslisted in FIG. 12 can also be detected by one or more systems disclosedherein, for distinguishing between adenoma with low grade dysplasia andadenoma with high grade dysplasia. For example, a detection system cancomprise one or more probes to detect one or more specific biomarkersfor distinguishing between adenoma with low grade dysplasia and adenomawith high grade dysplasia, such as listed in FIG. 12, of one or moreexosomes of a biological sample.

Ulcerative Colitis (UC) Versus Crohn's Disease (CD)

Ulcerative colitis (UC) versus Crohn's disease (CD) specific biomarkersfrom exosomes can include one or more (for example, 2, 3, 4, 5, 6, 7, 8,or more) overexpressed miRs, underexpressed miRs, mRNAs, geneticmutations, proteins, ligands, peptides, snoRNA, or any combinationthereof, such as listed in FIG. 13, and can be used to create a UCversus CD specific exosome bio-signature. For example, the one or moremRNAs that may be analyzed can include, but are not limited to, IFITM1,IFITM3, STAT1, STAT3, TAP1, PSME2, PSMB8, HNF4G, KLF5, AQP8, APT2B1,SLC16A, MFAP4, CCNG2, SLC44A4, DDAH1, TOB1, 231152_at, MKNK1, CEACAM7*,1562836_at, CDC42SE2, PSD3, 231169_at, IGL@*, GSN, GPM6B, CDV3*, PDPK1,ANP32E, ADAM9, CDH1, NLRP2, 215777_at, OSBPL1, VNN1, RABGAP1L, PHACTR2,ASH1L, 213710_s_at, CDH1, NLRP2, 215777_at, OSBPL1, VNN1, RABGAP1L,PHACTR2, ASH1, 213710_s_at, ZNF3, FUT2, IGHA1, EDEM1, GPR171, 229713_at,LOC643187, FLVCR1, SNAP23*, ETNK1, LOC728411, POSTN, MUC12, HOXA5,SIGLEC1, LARP5, PIGR, SPTBN1, UFM1, C6orf62, WDR90, ALDH1A3, F2RL1,IGHV1-69, DUOX2, RAB5A, or CP, or any combination thereof can also beused as specific biomarkers from exosomes for UC versus CD.

A biomarker mutation for distinguishing UC versus CD that can beassessed in an exosome includes, but is not limited to, a mutation ofCARD15, or any combination of mutations specific for distinguishing UCversus CD. The protein, ligand, or peptide that can be assessed in anexosome can include, but is not limited to, (P)ASCA.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between UC and CD, such as listedin FIG. 13. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more specific biomarkers fordistinguishing between UC and CD, such as listed in FIG. 13. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for having one or more specific biomarkers fordistinguishing between UC and CD, such as listed in FIG. 13.

One or more specific biomarkers for distinguishing between UC and CD,such as listed in FIG. 13 can also be detected by one or more systemsdisclosed herein, for distinguishing between UC and CD. For example, adetection system can comprise one or more probes to detect one or morespecific biomarkers for distinguishing between UC and CD, such as listedin FIG. 13, of one or more exosomes of a biological sample.

Hyperplastic Polyp

Hyperplastic polyp versus normal specific biomarkers from exosomes caninclude one or more (for example, 2, 3, 4, 5, 6, 7, 8, or more)overexpressed miRs, underexpressed miRs, mRNAs, genetic mutations,proteins, ligands, peptides, snoRNA, or any combination thereof, such aslisted in FIG. 14, and can be used to create a hyperplastic polyp versusnormal specific exosome bio-signature. For example, the one or moremRNAs that may be analyzed can include, but are not limited to, SLC6A14,ARHGEF10, ALS2, IL1RN, SPRy4, PTGER3, TRIM29, SERPINB5, 1560327 at, ZAK,BAG4, TRIB3, TTL, FOXQ1, or any combination.

Also provided herein is an isolated exosome comprising one or morehyperplastic polyp specific biomarkers, such as listed in FIG. 14. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more hyperplastic polyp specificbiomarkers, such as listed in FIG. 14. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for hyperplastic polyp specificexosomes or exosomes comprising one or more hyperplastic polyp specificbiomarkers, such as listed in FIG. 14.

One or more hyperplastic polyp specific biomarkers, such as listed inFIG. 14 can also be detected by one or more systems disclosed herein,for characterizing a hyperplastic polyp. For example, a detection systemcan comprise one or more probes to detect one or more listed in FIG. 14.One or more hyperplastic specific biomarkers, such as listed in FIG. 14,of one or more exosomes of a biological sample.

Adenoma with Low Grade Dysplasia Versus Normal

Adenoma with low grade dysplasia versus normal specific biomarkers fromexosomes can include one or more (for example, 2, 3, 4, 5, 6, 7, 8, ormore) overexpressed miRs, underexpressed miRs, mRNAs, genetic mutations,proteins, ligands, peptides, snoRNA, or any combination thereof, such aslisted in FIG. 15, and can be used to create an adenoma low gradedysplasia versus normal specific exosome bio-signature. For example, theRNAs that may be analyzed can include, but are not limited to, UGT2A3,KLK11, KIAA1199, FOXQ1, CLDN8, ABCA8, or PYY, or any combination thereofand can be used as specific biomarkers from exosomes for Adenoma lowgrade dysplasia versus normal. Furthermore, the snoRNA that can be usedas an exosomal biomarker for adenoma low grade dysplasia versus normalcan include, but is not limited to, GAS5.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between adenoma with low gradedysplasia and normal, such as listed in FIG. 15. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more specific biomarkers for distinguishing betweenadenoma with low grade dysplasia and normal, such as listed in FIG. 15.The composition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for having one or more specific biomarkers fordistinguishing between adenoma with low grade dysplasia and normal, suchas listed in FIG. 15.

One or more specific biomarkers for distinguishing between adenoma withlow grade dysplasia and normal, such as listed in FIG. 15 can also bedetected by one or more systems disclosed herein, for distinguishingbetween adenoma with low grade dysplasia and normal. For example, adetection system can comprise one or more probes to detect one or morespecific biomarkers for distinguishing between adenoma with low gradedysplasia and normal, such as listed in FIG. 15, of one or more exosomesof a biological sample.

Adenoma Versus Normal

Adenoma versus normal specific biomarkers from exosomes can include oneor more (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 16,and can be used to create an Adenoma versus normal specific exosomebio-signature. For example, the one or more mRNAs that may be analyzedcan include, but are not limited to, KIAA1199, FOXQ1, or CA7, or anycombination thereof. The protein, ligand, or peptide that can be used asa biomarker from exosomes that is specific to adenoma versus. normal caninclude, but is not limited to, Clusterin.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between adenoma and normal, suchas listed in FIG. 16. A composition comprising the isolated exosome isalso provided. Accordingly, in some embodiments, the compositioncomprises a population of exosomes comprising one or more specificbiomarkers for distinguishing between adenoma and normal, such as listedin FIG. 16. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for having one or more specific biomarkers fordistinguishing between adenoma and normal, such as listed in FIG. 16.

One or more specific biomarkers for distinguishing between adenoma andnormal, such as listed in FIG. 16 can also be detected by one or moresystems disclosed herein, for distinguishing between adenoma and normal.For example, a detection system can comprise one or more probes todetect one or more specific biomarkers for distinguishing betweenadenoma and normal, such as listed in FIG. 16, of one or more exosomesof a biological sample.

CRC Versus Normal

CRC versus normal specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 17,and can be used to create a CRC versus normal specific exosomebio-signature. For example, the one or mRNAs that may be analyzed caninclude, but are not limited to, VWF, IL8, CHI3L1, S100A8, GREM1, orODC, or any combination thereof and can be used as specific biomarkersfrom exosomes for CRC versus normal.

A biomarker mutation for CRC versus normal that can be assessed in anexosome includes, but is not limited to, a mutation of KRAS, BRAF, APC,MSH2, or MLH1, or any combination of mutations specific fordistinguishing between CRC versus normal. The protein, ligand, orpeptide that can be assessed in an exosome can include, but is notlimited to, cytokeratin 13, calcineurin, CHK1, clathrin light chain,phospho-ERK, phospho-PTK2, or MDM2, or any combination thereof.

Also provided herein is an isolated exosome comprising one or morespecific biomarkers for distinguishing between CRC and normal, such aslisted in FIG. 17. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more specific biomarkers fordistinguishing between CRC and normal, such as listed in FIG. 17. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for having one or more specific biomarkers fordistinguishing between CRC and normal, such as listed in FIG. 17.

One or more specific biomarkers for distinguishing between CRC andnormal, such as listed in FIG. 17 can also be detected by one or moresystems disclosed herein, for distinguishing between CRC and normal. Forexample, a detection system can comprise one or more probes to detectone or more specific biomarkers for distinguishing between CRC andnormal, such as listed in FIG. 17, of one or more exosomes of abiological sample.

Benign Prostatic Hyperplasia (BPH)

Benign prostatic hyperplasia (BPH) specific biomarkers from exosomes caninclude one or more (for example, 2, 3, 4, 5, 6, 7, 8, or more)overexpressed miRs, underexpressed miRs, mRNAs, genetic mutations,proteins, ligands, peptides, snoRNA, or any combination thereof, such aslisted in FIG. 18, and can be used to create a BPH specific exosomebio-signature. The protein, ligand, or peptide that can be assessed inan exosome can include, but is not limited to, intact fibronectin.

Also provided herein is an isolated exosome comprising one or more BPHspecific biomarkers, such as listed in FIG. 18 and in FIG. 1 for BPH. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more BPH specific biomarkers, such aslisted in FIG. 18 and in FIG. 1 for BPH. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for BPH specific exosomes orexosomes comprising one or more BPH specific biomarkers, such as listedin FIG. 18 and in FIG. 1 for BPH.

One or more BPH specific biomarkers, such as listed in FIG. 18 and inFIG. 1 for BPH, can also be detected by one or more systems disclosedherein, for characterizing a BPH. For example, a detection system cancomprise one or more probes to detect one more BPH specific biomarkers,such as listed in FIG. 18 and in FIG. 1 for BPH, of one or more exosomesof a biological sample.

Prostate Cancer

Prostate cancer specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 19,and can be used to create a prostate cancer specific exosomebio-signature. For example, a bio-signature for prostate cancer cancomprise miR-9, miR-21, miR-141, miR-370, miR-200b, miR-210, miR-155, ormiR-196a. In some embodiments, the bio-signature can comprise one ormore overexpressed miRs, such as, but not limited to, miR-202, miR-210,miR-296, miR-320, miR-370, miR-373, miR-498, miR-503, miR-184, miR-198,miR-302c, miR-345, miR-491, miR-513, miR-32, miR-182, miR-31,miR-26a-1/2, miR-200c, miR-375, miR-196a-1/2, miR-370, miR-425, miR-425,miR-194-1/2, miR-181a-1/2, miR-34b, let-71, miR-188, miR-25, miR-106b,miR-449, miR-99b, miR-93, miR-92-1/2, miR-125a, or miR-141, or anycombination thereof.

The bio-signature can also comprise one or more underexpressed miRs suchas, but not limited to, let-7a, let-7b, let-7c, let-7d, let-7g, miR-16,miR-23a, miR-23b, miR-26a, miR-92, miR-99a, miR-103, miR-125a, miR-125b,miR-143, miR-145, miR-195, miR-199, miR-221, miR-222, miR-497, let-7f,miR-19b, miR-22, miR-26b, miR-27a, miR-27b, miR-29a, miR-29b,miR-30_(—)5p, miR-30c, miR-100, miR-141, miR-148a, miR-205, miR-520h,miR-494, miR-490, miR-133a-1, miR-1-2, miR-218-2, miR-220, miR-128a,miR-221, miR-499, miR-329, miR-340, miR-345, miR-410, miR-126, miR-205,miR-7-1/2, miR-145, miR-34a, miR-487, or let-7b, or any combinationthereof. The bio-signature can comprise upregulated or overexpressedmiR-21, downregulated or underexpressed miR-15a, miR-16-1, miR-143 ormiR-145, or any combination thereof.

The one or more mRNAs that may be analyzed can include, but are notlimited to, AR, PCA3, or any combination thereof and can be used asspecific biomarkers from exosomes for prostate cancer.

The protein, ligand, or peptide that can be assessed in an exosome caninclude, but is not limited to, FASLG or TNFSF10 or any combinationthereof. Furthermore, an exosome isolated or assayed can be prostatecancer cell specific, or derived from prostate cancer cells.Furthermore, the snoRNA that can be used as an exosomal biomarker forprostate cancer can include, but is not limited to, U50. Examples ofprostate cancer bio-signatures are further described below.

Also provided herein is an isolated exosome comprising one or moreprostate cancer specific biomarkers, such as ACSL3-ETV1, C15ORF21-ETV1,FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5,SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4, or those listed inFIGS. 19, 60 and in FIG. 1 for prostate cancer. A composition comprisingthe isolated exosome is also provided. Accordingly, in some embodiments,the composition comprises a population of exosomes comprising one ormore prostate cancer specific biomarkers such as ACSL3-ETV1,C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5,TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4, orthose listed in FIGS. 19, 60 and in FIG. 1 for prostate cancer. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for prostate cancer specific exosomes or exosomes comprisingone or more prostate cancer specific biomarkers, such as ACSL3-ETV1,C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5,TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4, orthose listed in FIGS. 19, 60 and in FIG. 1 for prostate cancer.

One or more prostate cancer specific biomarkers, such as ACSL3-ETV1,C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5,TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4, orthose listed in FIGS. 19, 60 and in FIG. 1 for prostate cancer can alsobe detected by one or more systems disclosed herein, for characterizinga prostate cancer. For example, a detection system can comprise one ormore probes to detect one or more prostate cancer specific biomarkers,such as ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1,TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1,SLC5A3-ETV5 or KLK2-ETV4, or those listed in FIGS. 19, 60 and in FIG. 1for prostate cancer, of one or more exosomes of a biological sample.

Melanoma

Melanoma specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 20,and can be used to create a melanoma specific exosome bio-signature. Forexample, the bio-signature can comprise one or more overexpressed miRs,such as, but not limited to, miR-19a, miR-144, miR-200c, miR-211,miR-324-5p, miR-331, or miR-374, or any combination thereof. Thebio-signature can also comprise one or more underexpressed miRs such as,but not limited to, miR-9, miR-15a, miR-17-3p, miR-23b, miR-27a, miR-28,miR-29b, miR-30b, miR-31, miR-34b, miR-34c, miR-95, miR-96, miR-100,miR-104, miR-105, miR-106a, miR-107, miR-122a, miR-124a, miR-125b,miR-127, miR-128a, miR-128b, miR-129, miR-135a, miR-135b, miR-137,miR-138, miR-139, miR-140, miR-141, miR-149, miR-154, miR-154#3,miR-181a, miR-182, miR-183, miR-184, miR-185, miR-189, miR-190, miR-199,miR-199b, miR-200a, miR-200b, miR-204, miR-213, miR-215, miR-216,miR-219, miR-222, miR-224, miR-299, miR-302a, miR-302b, miR-302c,miR-302d, miR-323, miR-325, let-7a, let-7b, let-7d, let-7e, or let-7g,or any combination thereof.

The one or more mRNAs that may be analyzed can include, but are notlimited to, MUM-1, beta-catenin, or Nop/5/Sik, or any combinationthereof and can be used as specific biomarkers from exosomes formelanoma.

A biomarker mutation for melanoma that can be assessed in an exosomeincludes, but is not limited to, a mutation of CDK4 or any combinationof mutations specific for melanoma. The protein, ligand, or peptide thatcan be assessed in an exosome can include, but is not limited to,DUSP-1, Alix, hsp70, Gib2, Gia, moesin, GAPDH, malate dehydrogenase,p120 catenin, PGRL, syntaxin-binding protein 1 & 2, septin-2, orWD-repeat containing protein 1, or any combination thereof. The snoRNAthat can be used as an exosomal biomarker for melanoma include, but arenot limited to, H/ACA (U1071), SNORA11D, or any combination thereof.Furthermore, an exosome isolated or assayed can be melanoma cellspecific, or derived from melanoma cells.

Also provided herein is an isolated exosome comprising one or moremelanoma specific biomarkers, such as listed in FIG. 20 and in FIG. 1for melanoma. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more melanoma specificbiomarkers, such as listed in FIG. 20 and in FIG. 1 for melanoma. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for melanoma specific exosomes or exosomes comprising one ormore melanoma specific biomarkers, such as listed in FIG. 20 and in FIG.1 for melanoma.

One or more melanoma specific biomarkers, such as listed in FIG. 20 andin FIG. 1 for melanoma can also be detected by one or more systemsdisclosed herein, for characterizing a melanoma. For example, adetection system can comprise one or more probes to detect one or morecancer specific biomarkers, such as listed in FIG. 20 and in FIG. 1 formelanoma, of one or more exosomes of a biological sample.

Pancreatic Cancer

Pancreatic cancer specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 21,and can be used to create a pancreatic cancer specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-221, miR-181a,miR-155, miR-210, miR-213, miR-181b, miR-222, miR-181b-2, miR-21,miR-181b-1, miR-220, miR-181d, miR-223, miR-100-1/2, miR-125a, miR-143,miR-10a, miR-146, miR-99, miR-100, miR-199a-1, miR-10b, miR-199a-2,miR-221, miR-181a, miR-155, miR-210, miR-213, miR-181b, miR-222,miR-181b-2, miR-21, miR-181b-1, miR-181c, miR-220, miR-181d, miR-223,miR-100-1/2, miR-125a, miR-143, miR-10a, miR-146, miR-99, miR-100,miR-199a-1, miR-10b, miR-199a-2, miR-107, miR-103, miR-103-2,miR-125b-1, miR-205, miR-23a, miR-221, miR-424, miR-301, miR-100,miR-376a, miR-125b-1, miR-21, miR-16-1, miR-181a, miR-181c, miR-92,miR-15, miR-155, let-7f-1, miR-212, miR-107, miR-024-1/2, miR-18a,miR-31, miR-93, miR-224, or let-7d, or any combination thereof.

The bio-signature can also comprise one or more underexpressed miRs suchas, but not limited to, miR-148a, miR-148b, miR-375, miR-345, miR-142,miR-133a, miR-216, miR-217 or miR-139, or any combination thereof. Theone or more mRNAs that may be analyzed can include, but are not limitedto, PSCA, Mesothelin, or Osteopontin, or any combination thereof and canbe used as specific biomarkers from exosomes for pancreatic cancer.

A biomarker mutation for pancreatic cancer that can be assessed in anexosome includes, but is not limited to, a mutation of KRAS, CTNNLB1,AKT, NCOA3, or B-RAF, or any combination of mutations specific forpancreatic cancer. The biomarker can also be BRCA2, PALB2, or p16.Furthermore, an exosome isolated or assayed can be pancreatic cancercell specific, or derived from pancreatic cancer cells.

Also provided herein is an isolated exosome comprising one or morepancreatic cancer specific biomarkers, such as listed in FIG. 21. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more pancreatic cancer specificbiomarkers, such as listed in FIG. 21. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for pancreatic cancer specificexosomes or exosomes comprising one or more pancreatic cancer specificbiomarkers, such as listed in FIG. 21.

One or more pancreatic cancer specific biomarkers, such as listed inFIG. 21, can also be detected by one or more systems disclosed herein,for characterizing a pancreatic cancer. For example, a detection systemcan comprise one or more probes to detect one or more pancreatic cancerspecific biomarkers, such as listed in FIG. 21, of one or more exosomesof a biological sample.

Brain Cancer

Brain cancer (including, but not limited to, gliomas, glioblastomas,meinigiomas, acoustic neuroma/schwannomas, medulloblastoma) specificbiomarkers from exosomes can include one or more (for example, 2, 3, 4,5, 6, 7, 8, or more) overexpressed miRs, underexpressed miRs, mRNAs,genetic mutations, proteins, ligands, peptides, snoRNA, or anycombination thereof, such as listed in FIG. 22, and can be used tocreate a brain cancer specific exosome bio-signature. For example, thebio-signature can comprise one or more overexpressed miRs, such as, butnot limited to miR-21, miR-10b, miR-130a, miR-221, miR-125b-1,miR-125b-2, miR-9-2, miR-21, miR-25, or miR-123, or any combinationthereof.

The bio-signature can also comprise one or more underexpressed miRs suchas, but not limited to, miR-128a, miR-181c, miR-181a, or miR-181b, orany combination thereof. The one or more mRNAs that may be analyzedinclude, but are not limited to, MGMT, which can be used as specificbiomarker from exosomes for brain cancer. The protein, ligand, orpeptide that can be assessed in an exosome can include, but is notlimited to, EGFR.

Also provided herein is an isolated exosome comprising one or more braincancer specific biomarkers, such as GOPC-ROS1, or those listed in FIG.22 and in FIG. 1 for brain cancer. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebrain cancer specific biomarkers, such as GOPC-ROS1, or those listed inFIG. 22 and in FIG. 1 for brain cancer. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for brain cancer specific exosomesor exosomes comprising one or more brain cancer specific biomarkers,such as GOPC-ROS1, or those listed in FIG. 22. and in FIG. 1 for braincancer.

One or more brain cancer specific biomarkers, such as listed in FIG. 22and in FIG. 1 for brain cancer, can also be detected by one or moresystems disclosed herein, for characterizing a brain cancer. Forexample, a detection system can comprise one or more probes to detectone or more brain cancer specific biomarkers, such as GOPC-ROS1, orthose listed in FIG. 22 and in FIG. 1 for brain cancer, of one or moreexosomes of a biological sample.

Psoriasis

Psoriasis specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 23,and can be used to create a psoriasis specific exosome bio-signature.For example, the bio-signature can comprise one or more overexpressedmiRs, such as, but not limited to, miR-146b, miR-20a, miR-146a, miR-31,miR-200a, miR-17-5p, miR-30e-5p, miR-141, miR-203, miR-142-3p, miR-21,or miR-106a, or any combination thereof. The bio-signature can alsocomprise one or more underexpressed miRs such a, but not limited to,miR-125b, miR-99b, miR-122a, miR-197, miR-100, miR-381, miR-518b,miR-524, let-7e, miR-30c, miR-365, miR-133b, miR-10a, miR-133a, miR-22,miR-326, or miR-215, or any combination thereof.

The one or more mRNAs that may be analyzed can include, but are notlimited to, IL-20, VEGFR-1, VEGFR-2, VEGFR-3, or EGR1, or anycombination thereof and can be used as specific biomarkers from exosomesfor psoriasis. A biomarker mutation for psoriasis that can be assessedin an exosome includes, but is not limited to, a mutation of MGST2, orany combination of mutations specific for psoriasis.

Also provided herein is an isolated exosome comprising one or morepsoriasis specific biomarkers, such as listed in FIG. 23 and in FIG. 1for psoriasis. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more psoriasis specificbiomarkers, such as listed in FIG. 23 and in FIG. 1 for psoriasis. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for psoriasis specific exosomes or exosomes comprising oneor more psoriasis specific biomarkers, such as listed in FIG. 23 and inFIG. 1 for psoriasis.

One or more psoriasis specific biomarkers, such as listed in FIG. 23 andin FIG. 1 for psoriasis, can also be detected by one or more systemsdisclosed herein, for characterizing psoriasis. For example, a detectionsystem can comprise one or more probes to detect one or more psoriasisspecific biomarkers, such as listed in FIG. 23 and in FIG. 1 forpsoriasis, of one or more exosomes of a biological sample.

Cardiovascular Disease (CVD)

CVD specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 24,and can be used to create a CVD specific exosome bio-signature. Forexample, the bio-signature can comprise one or more overexpressed miRs,such as, but not limited to, miR-195, miR-208, miR-214, let-7b, let-7c,let-7e, miR-15b, miR-23a, miR-24, miR-27a, miR-27b, miR-93, miR-99b,miR-100, miR-103, miR-125b, miR-140, miR-145, miR-181a, miR-191,miR-195, miR-199a, miR-320, miR-342, miR-451, or miR-499, or anycombination thereof.

The bio-signature can also comprise one or more underexpressed miRs suchas, but not limited to, miR-1, miR-10a, miR-17-5p, miR-19a, miR-19b,miR-20a, miR-20b, miR-26b, miR-28, miR-30e-5p, miR-101, miR-106a,miR-126, miR-222, miR-374, miR-422b, or miR-423, or any combinationthereof. The mRNAs that may be analyzed can include, but are not limitedto, MRP14, CD69, or any combination thereof and can be used as specificbiomarkers from exosomes for CVD.

A biomarker mutation for CVD that can be assessed in an exosomeincludes, but is not limited to, a mutation of MYH7, SCN5A, or CHRM2, orany combination of mutations specific for CVD.

The protein, ligand, or peptide that can be assessed in an exosome caninclude, but is not limited to, CK-MB, cTnI (cardiac troponin), CRP,BPN, IL-6, MCSF, CD40, CD40L, or any combination thereof. Furthermore,an exosome isolated or assayed can be a CVD cell specific, or derivedfrom cardiac cells.

Also provided herein is an isolated exosome comprising one or more CVDspecific biomarkers, such as listed in FIG. 24 and in FIG. 1 for CVD. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more CVD specific biomarkers, such aslisted in FIG. 24 and in FIG. 1 for CVD. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for CVD specific exosomes orexosomes comprising one or more CVD specific biomarkers, such as listedin FIG. 24 and in FIG. 1 for CVD.

One or more CVD specific biomarkers, such as listed in FIG. 24 and inFIG. 1 for CVD, can also be detected by one or more system's disclosedherein, for characterizing a CVD. For example, a detection system cancomprise one or more probes to detect one or more CVD specificbiomarkers, such as listed in FIG. 24 and in FIG. 1 for CVD, of one ormore exosomes of a biological sample.

Blood Cancers

Hematological malignancies specific biomarkers from exosomes can includeone or more (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressedmiRs, underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 25,and can be used to create a hematological malignancies specific exosomebio-signature. For example, the one or more mRNAs that may be analyzedcan include, but are not limited to, HOX11, TAL1, LY1, LMO1, or LMO2, orany combination thereof and can be used as specific biomarkers fromexosomes for hematological malignancies.

A biomarker mutation for a blood cancer that can be assessed in anexosome includes, but is not limited to, a mutation of c-kit, PDGFR, orABL, or any combination of mutations specific for hematologicalmalignancies.

Also provided herein is an isolated exosome comprising one or more bloodcancer specific biomarkers, such as listed in FIG. 25 and in FIG. 1 forblood cancer. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more blood cancer specificbiomarkers, such as listed in FIG. 25 and in FIG. 1 for blood cancer.The composition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for blood cancer specific exosomes or exosomes comprisingone or more blood cancer specific biomarkers, such as listed in FIG. 25and in FIG. 1 for blood cancer.

One or more blood cancer specific biomarkers, such as listed in FIG. 25and in FIG. 1 for blood cancer, can also be detected by one or moresystems disclosed herein, for characterizing a blood cancer. Forexample, a detection system can comprise one or more probes to detectone or more blood cancer specific biomarkers, such as listed in FIG. 25and in FIG. 1 for blood cancer, of one or more exosomes of a biologicalsample.

The one or more blood cancer specific biomarkers can also be a genefusion selected from the group consisting of: TTL-ETV6, CDK6-MLL,CDK6-TLX3, ETV6-FLT3, ETV6-RUNX1, ETV6-TTL, MLL-AFF1, MLL-AFF3,MLL-AFF4, MLL-GAS7, TCBA1-ETV6, TCF3-PBX1 or TCF3-TFPT, for acutelymphocytic leukemia (ALL); BCL11B-TLX3, IL2-TNFRFS17, NUP214-ABL1,NUP98-CCDC28A, TAL1-STIL, or ETV6-ABL2, for T-cell acute lymphocyticleukemia (T-ALL); ATIC-ALK, KIAA1618-ALK, MSN-ALK, MYH9-ALK, NPM1-ALK,TGF-ALK or TPM3-ALK, for anaplastic large cell lymphoma (ALCL);BCR-ABL1, BCR-JAK2, ETV6-EVI1, ETV6-MN1 or ETV6-TCBA1, for chronicmyelogenous leukemia (CML); CBFB-MYH11, CHIC2-ETV6, ETV6-ABL1,ETV6-ABL2, ETV6-ARNT, ETV6-CDX2, ETV6-HLXB9, ETV6-PER1, MEF2D-DAZAP1,AML-AFF1, MLL-ARHGAP26, MLL-ARHGEF12, MLL-CASC5, MLL-CBL, MLL-CREBBP,MLL-DAB21P, MLL-ELL, MLL-EP300, MLL-EPS15, MLL-FNBP1, MLL-FOXO3A,MLL-GMPS, MLL-GPHN, MLL-MLLT1, MLL-MLLT11, MLL-MLLT3, MLL-MLLT6,MLL-MYO1F, MLL-PICALM, MLL-SEPT2, MLL-SEPT6, MLL-SORBS2, MYST3-SORBS2,MYST-CREBBP, NPM1-MLF1, NUP98-HOXA13, PRDM16-EVI1, RABEP1-PDGFRB,RUNX1-EVI1, RUNX1-MDS1, RUNX1-RPL22, RUNX1-RUNX1T1, RUNX1-SH3D19,RUNX1-USP42, RUNX1-YTHDF2, RUNX1-ZNF687, or TAF15-ZNF-384, for AML;CCND1-FSTL3, for chronic lymphocytic leukemia (CLL); and FLIP1-PDGFRA,FLT3-ETV6, KIAA1509-PDGFRA, PDE4DIP-PDGFRB, NIN-PDGFRB, TP53BP1-PDGFRB,or TPM3-PDGFRB, for hyper eosinophilia/chronic eosinophilia.

The one or more biomarkers for CLL can also include one or more of thefollowing upregulated or overexpressed miRNAs, such as miR-23b,miR-24-1, miR-146, miR-155, miR-195, miR-221, miR-331, miR-29a, miR-195,miR-34a, or miR-29c; one or more of the following downregulated orunderexpressed miRs, such as miR-15a, miR-16-1, miR-29 or miR-223, orany combination thereof.

The one or more biomarkers for ALL can also include one or more of thefollowing upregulated or overexpressed miRNAs, such as miR-128b,miR-204, miR-218, miR-331, miR-181b-1, miR-17-92; or any combinationthereof.

B-Cell Chronic Lymphocytic Leukemia (B-CLL)

B-CLL specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 26,and can be used to create a B-CLL specific exosome bio-signature. Forexample, the bio-signature can comprise one or more overexpressed miRs,such as, but not limited to, miR-183-prec, miR-190, miR-24-1-prec,miR-33, miR-19a, miR-140, miR-123, miR-10b, miR-15b-prec, miR-92-1,miR-188, miR-154, miR-217, miR-101, miR-141-prec, miR-153-prec,miR-196-2, miR-134, miR-141, miR-132, miR-192, or miR-181b-prec, or anycombination thereof.

The bio-signature can also comprise one or more underexpressed miRs suchas, but not limited to, miR-213, miR-220, or any combination thereof.The one or more mRNAs that may be analyzed can include, but are notlimited to, ZAP70, AdipoR1, or any combination thereof and can be usedas specific biomarkers from exosomes for B-CLL. A biomarker mutation forB-CLL that can be assessed in an exosome includes, but is not limitedto, a mutation of IGHV, P53, ATM, or any combination of mutationsspecific for B-CLL.

Also provided herein is an isolated exosome comprising one or more B-CLLspecific biomarkers, such as BCL3-MYC, MYC-BTG1, BCL7A-MYC,BRWD3-ARHGAP20 or BTG1-MYC, or those listed in FIG. 26. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more B-CLL specific biomarkers, such as BCL3-MYC,MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC, or those listed in FIG.26. The composition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for B-CLL specific exosomes or exosomes comprising one ormore B-CLL specific biomarkers, such as BCL3-MYC, MYC-BTG1, BCL7A-MYC,BRWD3-ARHGAP20 or BTG1-MYC, or those listed in FIG. 26.

One or more B-CLL specific biomarkers, such as BCL3-MYC, MYC-BTG1,BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC, or those listed in FIG. 26, canalso be detected by one or more systems disclosed herein, forcharacterizing a B-CLL. For example, a detection system can comprise oneor more probes to detect one or more B-CLL specific biomarkers, such asBCL3-MYC, MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC, or thoselisted in FIG. 26, of one or more exosomes of a biological sample.

B-Cell Lymphoma

B-cell lymphome specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 27,and can be used to create a B-cell lymphoma specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-17-92 polycistron,miR-155, miR-210, or miR-21, miR-19a, miR-92, miR-142 miR-155, miR-221miR-17-92, miR-21, miR-191, miR-205, or any combination thereof.Furthermore the snoRNA that can be used as an exosomal biomarker forB-cell lymphoma can include, but is not limited to, U50.

Also provided herein is an isolated exosome comprising one or moreB-cell lymphoma specific biomarkers, such as listed in FIG. 27. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more B-cell lymphoma specific biomarkers,such as listed in FIG. 27. The composition can comprise a substantiallyenriched population of exosomes, wherein the population of exosomes issubstantially homogeneous for B-cell lymphoma specific exosomes orexosomes comprising one or more B-cell lymphoma specific biomarkers,such as listed in FIG. 27.

One or more B-cell lymphoma specific biomarkers, such as listed in FIG.27, can also be detected by one or more systems disclosed herein, forcharacterizing a B-cell lymphoma. For example, a detection system cancomprise one or more probes to detect one or more B-cell lymphomaspecific biomarkers, such as listed in FIG. 27, of one or more exosomesof a biological sample.

Diffuse Large B-Cell Lymphoma (DLBCL)

DLBCL specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 28,and can be used to create a DLBCL specific exosome bio-signature. Forexample, the bio-signature can comprise one or more overexpressed miRs,such as, but not limited to, miR-17-92, miR-155, miR-210, or miR-21, orany combination thereof. The one or more mRNAs that may be analyzed caninclude, but are not limited to, A-myb, LMO2, JNK3, CD10, bcl-6, CyclinD2, IRF4, Flip, or CD44, or any combination thereof and can be used asspecific biomarkers from exosomes for DLBCL.

Also provided herein is an isolated exosome comprising one or more DLBCLspecific biomarkers, such as CITTA-BCL6, CLTC-ALK, IL21R-BCL6,PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK, or those listed in FIG.28. A composition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more DLBCL specific biomarkers, such asCITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 orSEC31A-ALK, or those listed in FIG. 28. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for DLBCL specific exosomes orexosomes comprising one or more DLBCL specific biomarkers, such asCITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 orSEC31A-ALK, or those listed in FIG. 28.

One or more DLBCL specific biomarkers, such as CITTA-BCL6, CLTC-ALK,IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK, or thoselisted in FIG. 28, can also be detected by one or more systems disclosedherein, for characterizing a DLBCL. For example, a detection system cancomprise one or more probes to detect one or more DLBCL specificbiomarkers, such as CITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM 1-BCL6,TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK, or those listed in FIG. 28, of oneor more exosomes of a biological sample.

Burkitt's Lymphoma

Burkitt's lymphoma specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 29,and can be used to create a Burkitt's lymphoma specific exosomebio-signature. For example, the bio-signature can also comprise one ormore underexpressed miRs such as, but not limited to, pri-miR-155, orany combination thereof. The one or more mRNAs that may be analyzed caninclude, but are not limited to, MYC, TERT, NS, NP, MAZ, RCF3, BYSL,IDE3, CDC7, TCL1A, AUTS2, MYBL1, BMP7, ITPR3, CDC2, BACK2, TTK, MME,ALOX5, or TOP1, or any combination thereof and can be used as specificbiomarkers from exosomes for Burkitt's lymphoma. The protein, ligand, orpeptide that can be assessed in an exosome can include, but is notlimited to, BCL6, KI-67, or any combination thereof.

Also provided herein is an isolated exosome comprising one or moreBurkitt's lymphoma specific biomarkers, such as IGH-MYC, LCP1-BCL6, orthose listed in FIG. 29. A composition comprising the isolated exosomeis also provided. Accordingly, in some embodiments, the compositioncomprises a population of exosomes comprising one or more Burkitt'slymphoma specific biomarkers, such as IGH-MYC, LCP1-BCL6, or thoselisted in FIG. 29. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for Burkitt's lymphoma specific exosomes orexosomes comprising one or more Burkitt's lymphoma specific biomarkers,such as IGH-MYC, LCP1-BCL6, or those listed in FIG. 29.

One or more Burkitt's lymphoma specific biomarkers, such as IGH-MYC,LCP1-BCL6, or those listed in FIG. 29, can also be detected by one ormore systems disclosed herein, for characterizing a Burkitt's lymphoma.For example, a detection system can comprise one or more probes todetect one or more Burkitt's lymphoma specific biomarkers, such asIGH-MYC, LCP1-BCL6, or those listed in FIG. 29, of one or more exosomesof a biological sample.

Hepatocellular Carcinoma

Hepatocellular carcinoma specific biomarkers from exosomes can includeone or more (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressedmiRs, underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 30and can be used to create a hepatocellular carcinoma specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-221. Thebio-signature can also comprise one or more underexpressed miRs such as,but not limited to, let-7a-1, let-7a-2, let-7a-3, let-7b, let-7c,let-7d, let-7e, let-7f-2, let-fg, miR-122a, miR-124a-2, miR-130a,miR-132, miR-136, miR-141, miR-142, miR-143, miR-145, miR-146, miR-150,miR-155(BIC), miR-181a-1, miR-181a-2, miR-181c, miR-195, miR-199a-1-5p,miR-199a-2-5p, miR-199b, miR-200b, miR-214, miR-223, or pre-miR-594, orany combination thereof. The one or more mRNAs that may be analyzed caninclude, but are not limited to, FAT10.

The one or more biomarkers of a bio-signature can also be used tocharacterize hepatitis C virus-associated hepatocellular carcinoma. Theone or more biomarkers can be a miRNA, such as an overexpressed orunderexpressed miRNA. For example, the upregulated or overexpressedmiRNA can be miR-122, miR-100, or miR-10a and the downregulated miRNAcan be miR-198 or miR-145.

Also provided herein is an isolated exosome comprising one or morehepatocellular carcinoma specific biomarkers, such as listed in FIG. 30and in FIG. 1 for hepatocellular carcinoma. A composition comprising theisolated exosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morehepatocellular carcinoma specific biomarkers, such as listed in FIG. 30and in FIG. 1 for hepatocellular carcinoma. The composition can comprisea substantially enriched population of exosomes, wherein the populationof exosomes is substantially homogeneous for hepatocellular carcinomaspecific exosomes or exosomes comprising one or more hepatocellularcarcinoma specific biomarkers, such as listed in FIG. 30 and in FIG. 1for hepatocellular carcinoma.

One or more hepatocellular carcinoma specific biomarkers, such as listedin FIG. 30 and in FIG. 1 for hepatocellular carcinoma, can also bedetected by one or more systems disclosed herein, for characterizing ahepatocellular carcinoma. For example, a detection system can compriseone or more probes to detect one or more hepatocellular carcinomaspecific biomarkers, such as listed in FIG. 30 and in FIG. 1 forhepatocellular carcinoma, of one or more exosomes of a biologicalsample.

Cervical Cancer

Cervical cancer specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 31,and can be used to create a cervical cancer specific exosomebio-signature. For example, the one or more mRNAs that may be analyzedcan include, but are not limited to, HPV E6, HPV E7, or p53, or anycombination thereof and can be used as specific biomarkers from exosomesfor cervical cancer.

Also provided herein is an isolated exosome comprising one or morecervical cancer specific biomarkers, such as listed in FIG. 31 and inFIG. 1 for cervical cancer. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morecervical cancer specific biomarkers, such as listed in FIG. 31 and inFIG. 1 for cervical cancer. The composition can comprise a substantiallyenriched population of exosomes, wherein the population of exosomes issubstantially homogeneous for cervical cancer specific exosomes orexosomes comprising one or more cervical cancer specific biomarkers,such as listed in FIG. 31 and in FIG. 1 for cervical cancer.

One or more cervical cancer specific biomarkers, such as listed in FIG.31 and in FIG. 1 for cervical cancer, can also be detected by one ormore systems disclosed herein, for characterizing a cervical cancer. Forexample, a detection system can comprise one or more probes to detectone or more cervical cancer specific biomarkers, such as listed in FIG.31 and in FIG. 1 for cervical cancer, of one or more exosomes of abiological sample.

Endometrial Cancer

Endometrial cancer specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 32and can be used to create a endometrial cancer specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-185, miR-106a,miR-181a, miR-210, miR-423, miR-103, miR-107, or let-7c, or anycombination thereof. The bio-signature can also comprise one or moreunderexpressed miRs such as, but not limited to, miR-71, miR-221,miR-193, miR-152, or miR-30c, or any combination thereof.

A biomarker mutation for endometrial cancer that can be assessed in anexosome includes, but is not limited to, a mutation of PTEN, K-RAS,B-catenin, p53, Her2/neu, or any combination of mutations specific forendometrial cancer. The protein, ligand, or peptide that can be assessedin an exosome can include, but is not limited to, NLRP7, AlphaV Beta6integrin, or any combination thereof.

Also provided herein is an isolated exosome comprising one or moreendometrial cancer specific biomarkers, such as listed in FIG. 32 and inFIG. 1 for endometrial cancer. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or moreendometrial cancer specific biomarkers, such as listed in FIG. 32 and inFIG. 1 for endometrial cancer. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for endometrial cancer specificexosomes or exosomes comprising one or more endometrial cancer specificbiomarkers, such as listed in FIG. 32 and in FIG. 1 for endometrialcancer.

One or more endometrial cancer specific biomarkers, such as listed inFIG. 32 and in FIG. 1 for endometrial cancer, can also be detected byone or more systems disclosed herein, for characterizing a endometrialcancer. For example, a detection system can comprise one or more probesto detect one or more endometrial cancer specific biomarkers, such aslisted in FIG. 32 and in FIG. 1 for endometrial cancer, of one or moreexosomes of a biological sample.

Head and Neck Cancer

Head and neck cancer specific biomarkers from exosomes can include oneor more (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 33,and can be used to create a head and neck cancer specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-21, let-7, miR-18,miR-29c, miR-142-3p, miR-155, miR-146b, miR-205, or miR-21, or anycombination thereof. The bio-signature can also comprise one or moreunderexpressed miRs such as, but not limited to, miR-494. The one ormore mRNAs that may be analyzed include, but are not limited to, HPV E6,HPV E7, p53, IL-8, SAT, H3FA3, or EGFR, or any combination thereof andcan be used as specific biomarkers from exosomes for head and neckcancer.

A biomarker mutation for head and neck cancer that can be assessed in anexosome includes, but is not limited to, a mutation of GSTM1, GSTT1,GSTP1, OGG1, XRCC1, XPD, RAD51, EGFR, p53, or any combination ofmutations specific for head and neck cancer. The protein, ligand, orpeptide that can be assessed in an exosome can include, but is notlimited to, EGFR, EphB4, or EphB2, or any combination thereof.

Also provided herein is an isolated exosome comprising one or more headand neck cancer specific biomarkers, such as CHCHD7-PLAG1, CTNNB1-PLAG1,FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1, or TCEA1-PLAG1, or those listed inFIG. 33 and in FIG. 1 for head and neck cancer. A composition comprisingthe isolated exosome is also provided. Accordingly, in some embodiments,the composition comprises a population of exosomes comprising one ormore head and neck cancer specific biomarkers, such as CHCHD7-PLAG1,CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1, or TCEA1-PLAG1, orthose listed in FIG. 33 and in FIG. 1 for head and neck cancer. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for head and neck cancer specific exosomes or exosomescomprising one or more head and neck cancer specific biomarkers, such asCHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1, orTCEA1-PLAG1, or those listed in FIG. 33 and in FIG. 1 for head and neckcancer.

One or more head and neck cancer specific biomarkers, such as listed inFIG. 33 and in FIG. 1 for head and neck cancer, can also be detected byone or more systems disclosed herein, for characterizing a head and neckcancer. For example, a detection system can comprise one or more probesto detect one or more head and neck cancer specific biomarkers, such asCHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1, orTCEA1-PLAG1, or those listed in FIG. 33 and in FIG. 1 for head and neckcancer, of one or more exosomes of a biological sample.

Inflammatory Bowel Disease (IBD)

IBD specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 34,and can be used to create a IBD specific exosome bio-signature. The oneor more mRNAs that may be analyzed can include, but are not limited to,Trypsinogen IV, SERT, or any combination thereof and can be used asspecific biomarkers from exosomes for IBD.

A biomarker mutation for IBD that can be assessed in an exosome caninclude, but is not limited to, a mutation of CARD15 or any combinationof mutations specific for IBD. The protein, ligand, or peptide that canbe assessed in an exosome can include, but is not limited to, II-16,II-1beta, II-12, TNF-alpha, interferon gamma, 11-6, Rantes, MCP-1,Resistin, or 5-HT, or any combination thereof.

Also provided herein is an isolated exosome comprising one or more IBDspecific biomarkers, such as listed in FIG. 34 and in FIG. 1 for IBD. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more IBD specific biomarkers, such aslisted in FIG. 34 and in FIG. 1 for IBD. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for IBD specific exosomes orexosomes comprising one or more IBD specific biomarkers, such as listedin FIG. 34 and in FIG. 1 for IBD.

One or more IBD specific biomarkers, such as listed in FIG. 34 and inFIG. 1 for IBD, can also be detected by one or more systems disclosedherein, for characterizing a IBD. For example, a detection system cancomprise one or more probes to detect one or more IBD specificbiomarkers, such as listed in FIG. 34 and in FIG. 1 for IBD, of one ormore exosomes of a biological sample.

Diabetes

Diabetes specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 35,and can be used to create a diabetes specific exosome bio-signature. Forexample, the one or more mRNAs that may be analyzed can include, but arenot limited to, 11-8, CTSS, ITGB2, HLA-DRA, CD53, PLAG27, or MMP9, orany combination thereof and can be used as specific biomarkers fromexosomes for diabetes. The protein, ligand, or peptide that can beassessed in an exosome can include, but is not limited to, RBP4.

Also provided herein is an isolated exosome comprising one or morediabetes specific biomarkers, such as listed in FIG. 35 and in FIG. 1for diabetes. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more diabetes specificbiomarkers, such as listed in FIG. 35 and in FIG. 1 for diabetes. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for diabetes specific exosomes or exosomes comprising one ormore diabetes specific biomarkers, such as listed in FIG. 35 and in FIG.1 for diabetes.

One or more diabetes specific biomarkers, such as listed in FIG. 35 andin FIG. 1 for diabetes, can also be detected by one or more systemsdisclosed herein, for characterizing a diabetes. For example, adetection system can comprise one or more probes to detect one or morediabetes specific biomarkers, such as listed in FIG. 35 and in FIG. 1for diabetes, of one or more exosomes of a biological sample.

Barrett's Esophagus

Barrett's Esophagus specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 36,and can be used to create a Barrett's Esophagus specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-21, miR-143,miR-145, miR-194, or miR-215, or any combination thereof. The one ormore mRNAs that may be analyzed include, but are not limited to, S100A2,S100A4, or any combination thereof and can be used as specificbiomarkers from exosomes for Barrett's Esophagus.

A biomarker mutation for Barrett's Esophagus that can be assessed in anexosome includes, but is not limited to, a mutation of p53 or anycombination of mutations specific for Barrett's Esophagus. The protein,ligand, or peptide that can be assessed in an exosome can include, butis not limited to, p53, MUC1, MUC2, or any combination thereof.

Also provided herein is an isolated exosome comprising one or moreBarrett's Esophagus specific biomarkers, such as listed in FIG. 36 andin FIG. 1 for Barrett's Esophagus. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or moreBarrett's Esophagus specific biomarkers, such as listed in FIG. 36 andin FIG. 1 for Barrett's Esophagus. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for Barrett's Esophagus specificexosomes or exosomes comprising one or more Barrett's Esophagus specificbiomarkers, such as listed in FIG. 36 and in FIG. 1 for Barrett'sEsophagus.

One or more Barrett's Esophagus specific biomarkers, such as listed inFIG. 36 and in FIG. 1 for Barrett's Esophagus, can also be detected byone or more systems disclosed herein, for characterizing a Barrett'sEsophagus. For example, a detection system can comprise one or moreprobes to detect one or more Barrett's Esophagus specific biomarkers,such as listed in FIG. 36 and in FIG. 1 for Barrett's Esophagus, of oneor more exosomes of a biological sample.

Fibromyalgia

Fibromyalgia specific biomarkers from exosomes can include one or more(for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 37,and can be used to create a fibromyalgia specific exosome bio-signature.The one or more mRNAs that may be analyzed can include, but are notlimited to, NR2D which can be used as a specific biomarker from exosomesfor fibromyalgia.

Also provided herein is an isolated exosome comprising one or morefibromyalgia specific biomarkers, such as listed in FIG. 37 and in FIG.1 for fibromyalgia. A composition comprising the isolated exosome isalso provided. Accordingly, in some embodiments, the compositioncomprises a population of exosomes comprising one or more fibromyalgiaspecific biomarkers, such as listed in FIG. 37 and in FIG. 1 forfibromyalgia. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for fibromyalgia specific exosomes or exosomescomprising one or more fibromyalgia specific biomarkers, such as listedin FIG. 37 and in FIG. 1 for fibromyalgia.

One or more fibromyalgia specific biomarkers, such as listed in FIG. 37and in FIG. 1 for fibromyalgia, can also be detected by one or moresystems disclosed herein, for characterizing a fibromyalgia. Forexample, a detection system can comprise one or more probes to detectone or more fibromyalgia specific biomarkers, such as listed in FIG. 37and in FIG. 1 for fibromyalgia, of one or more exosomes of a biologicalsample.

Stroke

Stroke specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 38,and can be used to create a stroke specific exosome bio-signature. Forexample, the one or more mRNAs that may be analyzed can include, but arenot limited to, MMP9, S100-P, S100A12, SI00A9, coag factor V, ArginaseI,CA-IV, monocarboxylic acid transporter, ets-2, EIF2alpha, cytoskeletonassociated protein 4, N-formylpeptide receptor, Ribonuclease2,N-acetylneuraminate pyruvate lyase, BCL-6, or Glycogen phosphorylase, orany combination thereof and can be used as specific biomarkers fromexosomes for stroke.

Also provided herein is an isolated exosome comprising one or morestroke specific biomarkers, such as listed in FIG. 38 and in FIG. 1 forstroke. A composition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more stroke specific biomarkers, such aslisted in FIG. 38 and in FIG. 1 for stroke. The composition can comprisea substantially enriched population of exosomes, wherein the populationof exosomes is substantially homogeneous for stroke specific exosomes orexosomes comprising one or more stroke specific biomarkers, such aslisted in FIG. 38 and in FIG. 1 for stroke.

One or more stroke specific biomarkers, such as listed in FIG. 38 and inFIG. 1 for stroke, can also be detected by one or more systems disclosedherein, for characterizing a stroke. For example, a detection system cancomprise one or more probes to detect one or more stroke specificbiomarkers, such as listed in FIG. 38 and in FIG. 1 for stroke, of oneor more exosomes of a biological sample.

Multiple Sclerosis (MS)

MS specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 39,and can be used to create a MS specific exosome bio-signature. Forexample, the one or more mRNAs that may be analyzed can include, but arenot limited to, IL-6, IL-17, PAR-3, IL-17, T1/ST2, JunD, 5-LO, LTA4H,MBP, PLP, or alpha-beta crystallin, or any combination thereof and canbe used as specific biomarkers from exosomes for MS.

Also provided herein is an isolated exosome comprising one or more MSspecific biomarkers, such as listed in FIG. 39 and in FIG. 1 for MS. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more MS specific biomarkers, such aslisted in FIG. 39 and in FIG. 1 for MS. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for MS specific exosomes orexosomes comprising one or more MS specific biomarkers, such as listedin FIG. 39 and in FIG. 1 for MS.

One or more MS specific biomarkers, such as listed in FIG. 39 and inFIG. 1 for MS, can also be detected by one or more systems disclosedherein, for characterizing a MS. For example, a detection system cancomprise one or more probes to detect one or more MS specificbiomarkers, such as listed in FIG. 39 and in FIG. 1 for MS, of one ormore exosomes of a biological sample.

Parkinson's Disease

Parkinson's disease specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 40,and can be used to create a Parkinson's disease specific exosomebio-signature. For example, the bio-signature can include, but is notlimited to, one or more underexpressed miRs such as miR-133b. The one ormore mRNAs that may be analyzed can include, but are not limited toNurr1, BDNF, TrkB, gstm1, or 5100 beta, or any combination thereof andcan be used as specific biomarkers from exosomes for Parkinson'sdisease.

A biomarker mutation for Parkinson's disease that can be assessed in anexosome includes, but is not limited to, a mutation of FGF20,alpha-synuclein, FGF20, NDUFV2, FGF2, CALB1, B2M, or any combination ofmutations specific for Parkinson's disease. The protein, ligand, orpeptide that can be assessed in an exosome can include, but is notlimited to, apo-H, Ceruloplasmin, BDNF, IL-8, Beta2-microglobulin,apoAII, tau, ABeta1-42, DJ-1, or any combination thereof.

Also provided herein is an isolated exosome comprising one or moreParkinson's disease specific biomarkers, such as listed in FIG. 40 andin FIG. 1 for Parkinson's disease A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or moreParkinson's disease specific biomarkers, such as listed in FIG. 40 andin FIG. 1 for Parkinson's disease. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for Parkinson's disease specificexosomes or exosomes comprising one or more Parkinson's disease specificbiomarkers, such as listed in FIG. 40 and in FIG. 1 for Parkinson'sdisease.

One or more Parkinson's disease specific biomarkers, such as listed inFIG. 40 and in FIG. 1 for Parkinson's disease, can also be detected byone or more systems disclosed herein, for characterizing a Parkinson'sdisease. For example, a detection system can comprise one or more probesto detect one or more Parkinson's disease specific biomarkers, such aslisted in FIG. 40 and in FIG. 1 for Parkinson's disease, of one or moreexosomes of a biological sample.

Rheumatic Disease

Rheumatic disease specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 41,and can be used to create a rheumatic disease specific exosomebio-signature. For example, the bio-signature can also comprise one ormore underexpressed miRs such as, but not limited to, miR-146a, miR-155,miR-132, miR-16, or miR-181, or any combination thereof. The one or moremRNAs that may be analyzed can include, but are not limited to, HOXD10,HOXD11, HOXD13, CCL8, LIM homeobox2, or CENP-E, or any combinationthereof and can be used as specific biomarkers from exosomes forrheumatic disease. The protein, ligand, or peptide that can be assessedin an exosome can include, but is not limited to, TNFα.

Also provided herein is an isolated exosome comprising one or morerheumatic disease specific biomarkers, such as listed in FIG. 41 and inFIG. 1 for rheumatic disease. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morerheumatic disease specific biomarkers, such as listed in FIG. 41 and inFIG. 1 for rheumatic disease. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for rheumatic disease specificexosomes or exosomes comprising one or more rheumatic disease specificbiomarkers, such as listed in FIG. 41 and in FIG. 1 for rheumaticdisease.

One or more rheumatic disease specific biomarkers, such as listed inFIG. 41 and in FIG. 1 for rheumatic disease, can also be detected by oneor more systems disclosed herein, for characterizing a rheumaticdisease. For example, a detection system can comprise one or more probesto detect one or more rheumatic disease specific biomarkers, such aslisted in FIG. 41 and in FIG. 1 for rheumatic disease, of one or moreexosomes of a biological sample.

Alzheimer's Disease

Alzheimer's disease specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 42,and can be used to create a Alzheimers disease specific exosomebio-signature. For example, the bio-signature can also comprise one ormore underexpressed miRs such as miR-107, miR-29a, miR-29b-1, or miR-9,or any combination thereof. The bio-signature can also comprise one ormore overexpressed miRs such as miR-128 or any combination thereof.

The one or more mRNAs that may be analyzed can include, but are notlimited to, HIF-1α, BACE1, Reelin, CHRNA7, or 3Rtau/4Rtau, or anycombination thereof and can be used as specific biomarkers from exosomesfor Alzheimer's disease.

A biomarker mutation for Alzheimer's disease that can be assessed in anexosome includes, but is not limited to, a mutation of APP, presenilin1,presenilin2, APOE4, or any combination of mutations specific forAlzheimer's disease. The protein, ligand, or peptide that can beassessed in an exosome can include, but is not limited to, BACE1,Reelin, Cystatin C, Truncated Cystatin C, Amyloid Beta, C3a, t-Tau,Complement factor H, or alpha-2-macroglobulin, or any combinationthereof.

Also provided herein is an isolated exosome comprising one or moreAlzheimer's disease specific biomarkers, such as listed in FIG. 42 andin FIG. 1 for Alzheimer's disease. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or moreAlzheimer's disease specific biomarkers, such as listed in FIG. 42 andin FIG. 1 for Alzheimer's disease. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for Alzheimer's disease specificexosomes or exosomes comprising one or more Alzheimer's disease specificbiomarkers, such as listed in FIG. 42 and in FIG. 1 for Alzheimer'sdisease.

One or more Alzheimer's disease specific biomarkers, such as listed inFIG. 42 and in FIG. 1 for Alzheimer's disease, can also be detected byone or more systems disclosed herein, for characterizing a Alzheimer'sdisease. For example, a detection system can comprise one or more probesto detect one or more Alzheimer's disease specific biomarkers, such aslisted in FIG. 42 and in FIG. 1 for Alzheimer's disease, of one or moreexosomes of a biological sample.

Prion Disease

Prion specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 43,and can be used to create a prion specific exosome bio-signature. Forexample, the one or more mRNAs that may be analyzed can include, but arenot limited to, Amyloid B4, App, IL-1R1, or SOD1, or any combinationthereof and can be used as specific biomarkers from exosomes for aprion. The protein, ligand, or peptide that can be assessed in anexosome can include, but is not limited to, PrP(c), 14-3-3, NSE, S-100,Tau, AQP-4, or any combination thereof.

Also provided herein is an isolated exosome comprising one or more priondisease specific biomarkers, such as listed in FIG. 43 and in FIG. 1 forprion disease. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more prion disease specificbiomarkers, such as listed in FIG. 43 and in FIG. 1 for prion disease.The composition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for prion disease specific exosomes or exosomes comprisingone or more prion disease specific biomarkers, such as listed in FIG. 43and in FIG. 1 for prion disease.

One or more prion disease specific biomarkers, such as listed in FIG. 43and in FIG. 1 for prion disease, can also be detected by one or moresystems disclosed herein, for characterizing a prion disease. Forexample, a detection system can comprise one or more probes to detectone or more prion disease specific biomarkers, such as listed in FIG. 43and in FIG. 1 for prion disease, of one or more exosomes of a biologicalsample.

Sepsis

Sepsis specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 44,and can be used to create a sepsis specific exosome bio-signature. Forexample, the one or more mRNAs that may be analyzed can include, but arenot limited to, 15-Hydroxy-PG dehydrogenase (up), LAIR1 (up), NFKB1A(up), TLR2, PGLYPR1, TLR4, MD2, TLR5, IFNAR2, IRAK2, IRAK3, IRAK4, PI3K,PI3KCB, MAP2K6, MAPK14, NFKB1A, NFKB1, IL1R1, MAP2K1IP1, MKNK1, FAS,CASP4, GADD45B, SOCS3, TNFSF10, TNFSF13B, OSM, HGF, or IL18R1, or anycombination thereof and can be used as specific biomarkers from exosomesfor sepsis.

Also provided herein is an isolated exosome comprising one or moresepsis specific biomarkers, such as listed in FIG. 44. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more sepsis specific biomarkers, such as listed inFIG. 44. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for sepsis specific exosomes or exosomescomprising one or more sepsis specific biomarkers, such as listed inFIG. 44.

One or more sepsis specific biomarkers, such as listed in FIG. 44, canalso be detected by one or more systems disclosed herein, forcharacterizing a sepsis. For example, a detection system can compriseone or more probes to detect one or more sepsis specific biomarkers,such as listed in FIG. 44, of one or more exosomes of a biologicalsample.

Chronic Neuropathic Pain

Chronic neuropathic pain (CNP) specific biomarkers from exosomes caninclude one or more (for example, 2, 3, 4, 5, 6, 7, 8, or more)overexpressed miRs, underexpressed miRs, mRNAs, genetic mutations,proteins, ligands, peptides, snoRNA, or any combination thereof, such aslisted in FIG. 45, and can be used to create a CNP specific exosomebio-signature. For example, the one or more mRNAs that may be analyzedcan include, but are not limited to, ICAM-1 (rodent), CGRP (rodent),TIMP-1 (rodent), CLR-1 (rodent), HSP-27 (rodent), FABP (rodent), orapolipoprotein D (rodent), or any combination thereof and can be used asspecific biomarkers from exosomes for CNP. The protein, ligand, orpeptide that can be assessed in an exosome can include, but is notlimited to, chemokines, chemokine receptors (CCR2/4), or any combinationthereof.

Also provided herein is an isolated exosome comprising one or morechronic neuropathic pain specific biomarkers, such as listed in FIG. 45and in FIG. 1 for chronic neuropathic pain. A composition comprising theisolated exosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morechronic neuropathic pain specific biomarkers, such as listed in FIG. 45and in FIG. 1 for chronic neuropathic pain. The composition can comprisea substantially enriched population of exosomes, wherein the populationof exosomes is substantially homogeneous for chronic neuropathic painspecific exosomes or exosomes comprising one or more chronic neuropathicpain specific biomarkers, such as listed in FIG. 45 and in FIG. 1 forchronic neuropathic pain.

One or more chronic neuropathic pain specific biomarkers, such as listedin FIG. 45 and in FIG. 1 for chronic neuropathic pain, can also bedetected by one or more systems disclosed herein, for characterizing achronic neuropathic pain. For example, a detection system can compriseone or more probes to detect one or more chronic neuropathic painspecific biomarkers, such as listed in FIG. 45 and in FIG. 1 for chronicneuropathic pain, of one or more exosomes of a biological sample.

Peripheral Neuropathic Pain

Peripheral neuropathic pain (PNP) specific biomarkers from exosomes caninclude one or more (for example, 2, 3, 4, 5, 6, 7, 8, or more)overexpressed miRs, underexpressed miRs, mRNAs, genetic mutations,proteins, ligands, peptides, snoRNA, or any combination thereof, such aslisted in FIG. 46, and can be used to create a PNP specific exosomebio-signature. For example, the protein, ligand, or peptide that can beassessed in an exosome can include, but is not limited to, OX42, ED9, orany combination thereof.

Also provided herein is an isolated exosome comprising one or moreperipheral neuropathic pain specific biomarkers, such as listed in FIG.46 and in FIG. 1 for peripheral neuropathic pain. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more peripheral neuropathic pain specific biomarkers,such as listed in FIG. 46 and in FIG. 1 for peripheral neuropathic pain.The composition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for peripheral neuropathic pain specific exosomes orexosomes comprising one or more peripheral neuropathic pain specificbiomarkers, such as listed in FIG. 46 and in FIG. 1 for peripheralneuropathic pain.

One or more peripheral neuropathic pain specific biomarkers, such aslisted in FIG. 46 and in FIG. 1 for peripheral neuropathic pain, canalso be detected by one or more systems disclosed herein, forcharacterizing a peripheral neuropathic pain. For example, a detectionsystem can comprise one or more probes to detect one or more peripheralneuropathic pain specific biomarkers, such as listed in FIG. 46 and inFIG. 1 for peripheral neuropathic pain, of one or more exosomes of abiological sample.

Schizophrenia

Schizophrenia specific biomarkers from exosomes can include one or more(for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 47,and can be used to create a schizophrenia specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-181b. Thebio-signature can also comprise one or more underexpressed miRs such as,but not limited to, miR-7, miR-24, miR-26b, miR-29b, miR-30b, miR-30e,miR-92, or miR-195, or any combination thereof.

The one or more mRNAs that may be analyzed can include, but are notlimited to, IFITM3, SERPINA3, GLS, or ALDH7A1BASP1, or any combinationthereof and can be used as specific biomarkers from exosomes forschizophrenia. A biomarker mutation for schizophrenia that can beassessed in an exosome includes, but is not limited to, a mutation of toDISC1, dysbindin, neuregulin-1, seratonin 2a receptor, NURR1, or anycombination of mutations specific for schizophrenia.

The protein, ligand, or peptide that can be assessed in an exosome caninclude, but is not limited to, ATP5B, ATP5H, ATP6V1B, DNM1, NDUFV2,NSF, PDHB, or any combination thereof.

Also provided herein is an isolated exosome comprising one or moreschizophrenia specific biomarkers, such as listed in FIG. 47 and in FIG.1 for schizophrenia. A composition comprising the isolated exosome isalso provided. Accordingly, in some embodiments, the compositioncomprises a population of exosomes comprising one or more schizophreniaspecific biomarkers, such as listed in FIG. 47 and in FIG. 1 forschizophrenia. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for schizophrenia specific exosomes orexosomes comprising one or more schizophrenia specific biomarkers, suchas listed in FIG. 47 and in FIG. 1 for schizophrenia.

One or more schizophrenia specific biomarkers, such as listed in FIG. 47and in FIG. 1 for schizophrenia, can also be detected by one or moresystems disclosed herein, for characterizing a schizophrenia. Forexample, a detection system can comprise one or more probes to detectone or more schizophrenia specific biomarkers, such as listed in FIG. 47and in FIG. 1 for schizophrenia, of one or more exosomes of a biologicalsample.

Bipolar Disease

Bipolar disease specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 48,and can be used to create a bipolar disease specific exosomebio-signature. For example, the one or more mRNAs that may be analyzedcan include, but are not limited to, FGF2, ALDH7A1, AGXT2L1, AQP4, orPCNT2, or any combination thereof and can be used as specific biomarkersfrom exosomes for bipolar disease. A biomarker mutation for bipolardisease that can be assessed in an exosome includes, but is not limitedto, a mutation of Dysbindin, DAOA/G30, DISC1, neuregulin-1, or anycombination of mutations specific for bipolar disease.

Also provided herein is an isolated exosome comprising one or morebipolar disease specific biomarkers, such as listed in FIG. 48. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more bipolar disease specific biomarkers,such as listed in FIG. 48. The composition can comprise a substantiallyenriched population of exosomes, wherein the population of exosomes issubstantially homogeneous for bipolar disease specific exosomes orexosomes comprising one or more bipolar disease specific biomarkers,such as listed in FIG. 48.

One or more bipolar disease specific biomarkers, such as listed in FIG.48, can also be detected by one or more systems disclosed herein, forcharacterizing a bipolar disease. For example, a detection system cancomprise one or more probes to detect one or more bipolar diseasespecific biomarkers, such as listed in FIG. 48, of one or more exosomesof a biological sample.

Depression

Depression specific biomarkers from exosomes can include one or more(for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 49,and can be used to create a depression specific exosome bio-signature.For example, the one or more mRNAs that may be analyzed can include, butare not limited to, FGFR1, FGFR2, FGFR3, or AQP4, or any combinationthereof can also be used as specific biomarkers from exosomes fordepression.

Also provided herein is an isolated exosome comprising one or moredepression specific biomarkers, such as listed in FIG. 49. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more depression specific biomarkers, such as listed inFIG. 49. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for depression specific exosomes or exosomescomprising one or more depression specific biomarkers, such as listed inFIG. 49.

One or more depression specific biomarkers, such as listed in FIG. 49,can also be detected by one or more systems disclosed herein, forcharacterizing a depression. For example, a detection system cancomprise one or more probes to detect one or more depression specificbiomarkers, such as listed in FIG. 49, of one or more exosomes of abiological sample.

Gastrointestinal Stromal Tumor (GIST)

GIST specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 50,and can be used to create a GIST specific exosome bio-signature. Forexample, the one or more mRNAs that may be analyzed can include, but arenot limited to, DOG-1, PKC-theta, KIT, GPR20, PRKCQ, KCNK3, KCNH2, SCG2,TNFRSF6B, or CD34, or any combination thereof and can be used asspecific biomarkers from exosomes for GIST.

A biomarker mutation for GIST that can be assessed in an exosomeincludes, but is not limited to, a mutation of PKC-theta or anycombination of mutations specific for GIST. The protein, ligand, orpeptide that can be assessed in an exosome can include, but is notlimited to, PDGFRA, c-kit, or any combination thereof.

Also provided herein is an isolated exosome comprising one or more GISTspecific biomarkers, such as listed in FIG. 50 and in FIG. 1 for GIST. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more GIST specific biomarkers, such aslisted in FIG. 50 and in FIG. 1 for GIST. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for GIST specific exosomes orexosomes comprising one or more GIST specific biomarkers, such as listedin FIG. 50 and in FIG. 1 for GIST.

One or more GIST specific biomarkers, such as listed in FIG. 50 and inFIG. 1 for GIST, can also be detected by one or more systems disclosedherein, for characterizing a GIST. For example, a detection system cancomprise one or more probes to detect one or more GIST specificbiomarkers, such as listed in FIG. 50 and in FIG. 1 for GIST, of one ormore exosomes of a biological sample.

Renal Cell Carcinoma

Renal cell carcinoma specific biomarkers from exosomes can include oneor more (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 51,and can be used to create a renal cell carcinoma specific exosomebio-signature. For example, the bio-signature can also comprise one ormore underexpressed miRs such as, but not limited to, miR-141, miR-200c,or any combination thereof. The one or more upregulated or overexpressedmiRNA can be miR-28, miR-185, miR-27, miR-let-7f-2, or any combinationthereof.

The one or more mRNAs that may be analyzed can include, but are notlimited to, laminin receptor 1, betaig-h3, Galectin-1, a-2Macroglobulin, Adipophilin, Angiopoietin 2, Caldesmon 1, Class IIMHC-associated invariant chain (CD74), Collagen IV-al, Complementcomponent, Complement component 3, Cytochrome P450, subfamily IIJpolypeptide 2, Delta sleep-inducing peptide, Fc g receptor 111a (CD16),HLA-B, HLA-DRa, HLA-DRb, HLA-SB, IFN-induced transmembrane protein 3,IFN-induced transmembrane protein 1, or Lysyl Oxidase, or anycombination thereof and can be used as specific biomarkers from exosomesfor renal cell carcinoma.

A biomarker mutation for renal cell carcinoma that can be assessed in anexosome includes, but is not limited to, a mutation of VHL or anycombination of mutations specific renal cell carcinoma.

The protein, ligand, or peptide that can be assessed in an exosome caninclude, but is not limited to, IF1 alpha, VEGF, PDGFRA, or anycombination thereof.

Also provided herein is an isolated exosome comprising one or more RCCspecific biomarkers, such as ALPHA-TFEB, NONO-TFE3, PRCC-TFE3,SFPQ-TFE3, CLTC-TFE3, or MALAT1-TFEB, or those listed in FIG. 51 and inFIG. 1 for RCC. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more RCC specific biomarkers,such as ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3, orMALAT1-TFE, or those listed in FIG. 51 and in FIG. 1 for RCC. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for RCC specific exosomes or exosomes comprising one or moreRCC specific biomarkers, such as ALPHA-TFEB, NONO-TFE3, PRCC-TFE3,SFPQ-TFE3, CLTC-TFE3, or MALAT1-TFE, or those listed in FIG. 51 and inFIG. 1 for RCC.

One or more RCC specific biomarkers, such as ALPHA-TFEB, NONO-TFE3,PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3, or MALAT1-TFE, or those listed in FIG.51 and in FIG. 1 for RCC, can also be detected by one or more systemsdisclosed herein, for characterizing a RCC. For example, a detectionsystem can comprise one or more probes to detect one or more RCCspecific biomarkers, such as ALPHA-TFEB, NONO-TFE3, PRCC-TFE3,SFPQ-TFE3, CLTC-TFE3, or MALAT1-TFE, or those listed in FIG. 51 and inFIG. 1 for RCC, of one or more exosomes of a biological sample.

Cirrhosis

Cirrhosis specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 52,and can be used to create a cirrhosis specific exosome bio-signature.The one or more mRNAs that may be analyzed include, but are not limitedto, NLT, which can be used as aspecific biomarker from exosomes forcirrhosis.

The protein, ligand, or peptide that can be assessed in an exosome caninclude, but is not limited to, NLT, HBsAG, AST, YKL-40, Hyaluronicacid, TIMP-1, alpha 2 macroglobulin, a-1-antitrypsin P1Z allele,haptoglobin, or acid phosphatase ACP AC, or any combination thereof.

Also provided herein is an isolated exosome comprising one or morecirrhosis specific biomarkers, such as those listed in FIG. 52 and inFIG. 1 for cirrhosis. A composition comprising the isolated exosome isalso provided. Accordingly, in some embodiments, the compositioncomprises a population of exosomes comprising one or more cirrhosisspecific biomarkers, such as those listed in FIG. 52 and in FIG. 1 forcirrhosis. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for cirrhosis specific exosomes or exosomescomprising one or more cirrhosis specific biomarkers, such as thoselisted in FIG. 52 and in FIG. 1 for cirrhosis.

One or more cirrhosis specific biomarkers, such as those listed in FIG.52 and in FIG. 1 for cirrhosis, can also be detected by one or moresystems disclosed herein, for characterizing cirrhosis. For example, adetection system can comprise one or more probes to detect one or morecirrhosis specific biomarkers, such as those listed in FIG. 52 and inFIG. 1 for cirrhosis, of one or more exosomes of a biological sample.

Esophageal Cancer

Esophageal cancer specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 53,and can be used to create a esophageal cancer specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-192, miR-194,miR-21, miR-200c, miR-93, miR-342, miR-152, miR-93, miR-25, miR-424, ormiR-151, or any combination thereof. The bio-signature can also compriseone or more underexpressed miRs such as, but not limited to, miR-27b,miR-205, miR-203, miR-342, let-7c, miR-125b, miR-100, miR-152, miR-192,miR-194, miR-27b, miR-205, miR-203, miR-200c, miR-99a, miR-29c, miR-140,miR-103, or miR-107, or any combination thereof. The one or more mRNAsthat may be analyzed include, but are not limited to, MTHFR and can beused as specific biomarkers from exosomes for esophageal cancer.

Also provided herein is an isolated exosome comprising one or moreesophageal cancer specific biomarkers, such as listed in FIG. 53 and inFIG. 1 for esophageal cancer. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or moreesophageal cancer specific biomarkers, such as listed in FIG. 53 and inFIG. 1 for esophageal cancer. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for esophageal cancer specificexosomes or exosomes comprising one or more esophageal cancer specificbiomarkers, such as listed in FIG. 53 and in FIG. 1 for esophagealcancer.

One or more esophageal cancer specific biomarkers, such as listed inFIG. 53 and in FIG. 1 for esophageal cancer, can also be detected by oneor more systems disclosed herein, for characterizing a esophagealcancer. For example, a detection system can comprise one or more probesto detect one or more esophageal cancer specific biomarkers, such aslisted in FIG. 53 and in FIG. 1 for esophageal cancer, of one or moreexosomes of a biological sample.

Gastric Cancer

Gastric cancer specific biomarkers from exosomes can include one or more(for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 54,and can be used to create a gastric cancer specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-106a, miR-21,miR-191, miR-223, miR-24-1, miR-24-2, miR-107, miR-92-2, miR-214,miR-25, or miR-221, or any combination thereof. The bio-signature canalso comprise one or more underexpressed miRs such as, but not limitedto, let-7a.

The one or more mRNAs that may be analyzed include, but are not limitedto, RRM2, EphA4, or survivin, or any combination thereof and can be usedas specific biomarkers from exosomes for gastric cancer. A biomarkermutation for gastric cancer that can be assessed in an exosome includes,but is not limited to, a mutation of APC or any combination of mutationsspecific for gastric cancer. The protein, ligand, or peptide that can beassessed in an exosome can include, but is not limited to EphA4.

Also provided herein is an isolated exosome comprising one or moregastric cancer specific biomarkers, such as listed in FIG. 54. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more gastric cancer specific biomarkers,such as listed in FIG. 54. The composition can comprise a substantiallyenriched population of exosomes, wherein the population of exosomes issubstantially homogeneous for gastric cancer specific exosomes orexosomes comprising one or more gastric cancer specific biomarkers, suchas listed in FIG. 54.

One or more gastric cancer specific biomarkers, such as listed in FIG.54, can also be detected by one or more systems disclosed herein, forcharacterizing a gastric cancer. For example, a detection system cancomprise one or more probes to detect one or more gastric cancerspecific biomarkers, such as listed in FIG. 54, of one or more exosomesof a biological sample.

Autism

Autism specific biomarkers from exosomes can include one or more (forexample, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 55,and can be used to create an autism specific exosome bio-signature. Forexample, the bio-signature can comprise one or more overexpressed miRs,such as, but not limited to, miR-484, miR-21, miR-212, miR-23a, miR-598,miR-95, miR-129, miR-431, miR-7, miR-15a, miR-27a, miR-15b, miR-148b,miR-132, or miR-128, or any combination thereof. The bio-signature canalso comprise one or more underexpressed miRs such as, but not limitedto, miR-93, miR-106a, miR-539, miR-652, miR-550, miR-432, miR-193b,miR-181d, miR-146b, miR-140, miR-381, miR-320a, or miR-106b, or anycombination thereof. The protein, ligand, or peptide that can beassessed in an exosome can include, but is not limited to, GM1, GD1a,GD1b, or GT1b, or any combination thereof.

Also provided herein is an isolated exosome comprising one or moreautism specific biomarkers, such as listed in FIG. 55 and in FIG. 1 forautism. A composition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more autism specific biomarkers, such aslisted in FIG. 55 and in FIG. 1 for autism. The composition can comprisea substantially enriched population of exosomes, wherein the populationof exosomes is substantially homogeneous for autism specific exosomes orexosomes comprising one or more autism specific biomarkers, such aslisted in FIG. 55 and in FIG. 1 for autism.

One or more autism specific biomarkers, such as listed in FIG. 55 and inFIG. 1 for autism, can also be detected by one or more systems disclosedherein, for characterizing a autism. For example, a detection system cancomprise one or more probes to detect one or more autism specificbiomarkers, such as listed in FIG. 55 and in FIG. 1 for autism, of oneor more exosomes of a biological sample.

Organ Rejection

Organ rejection specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 56,and can be used to create an organ rejection specific exosomebio-signature. For example, the bio-signature can comprise one or moreoverexpressed miRs, such as, but not limited to, miR-658, miR-125a,miR-320, miR-381, miR-628, miR-602, miR-629, or miR-125a, or anycombination thereof. The bio-signature can also comprise one or moreunderexpressed miRs such as, but not limited to, miR-324-3p, miR-611,miR-654, miR-330_MM1, miR-524, miR-17-3p_MM1, miR-483, miR-663,miR-5,6-5p, miR-326, miR-197_MM2, or miR-346, or any combinationthereof. The protein, ligand, or peptide that can be assessed in anexosome can include, but is not limited to, matix metalloprotein-9,proteinase 3, or HNP, or any combinations thereof. The biomarker can bea member of the matrix metalloproteinases.

Also provided herein is an isolated exosome comprising one or more organrejection specific biomarkers, such as listed in FIG. 56. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more organ rejection specific biomarkers, such aslisted in FIG. 56. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for organ rejection specific exosomes orexosomes comprising one or more organ rejection specific biomarkers,such as listed in FIG. 56.

One or more organ rejection specific biomarkers, such as listed in FIG.56, can also be detected by one or more systems disclosed herein, forcharacterizing a organ rejection. For example, a detection system cancomprise one or more probes to detect one or more organ rejectionspecific biomarkers, such as listed in FIG. 56, of one or more exosomesof a biological sample.

Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus specific biomarkers fromexosomes can include one or more (for example, 2, 3, 4, 5, 6, 7, 8, ormore) overexpressed miRs, underexpressed miRs, mRNAs, genetic mutations,proteins, ligands, peptides, snoRNA, or any combination thereof, such aslisted in FIG. 57, and can be used to create a methicillin-resistantStaphylococcus aureus specific exosome bio-signature.

The one or more mRNAs that may be analyzed include, but are not limitedto, TSST-1 which can be used as a specific biomarker from exosomes formethicillin-resistant Staphylococcus aureus. A biomarker mutation formethicillin-resistant Staphylococcus aureus that can be assessed in anexosome includes, but is not limited to, a mutation of mecA, Protein ASNPs, or any combination of mutations specific for methicillin-resistantStaphylococcus aureus. The protein, ligand, or peptide that can beassessed in an exosome can include, but is not limited to, ETA, ETB,TSST-1, or leukocidins, or any combination thereof.

Also provided herein is an isolated exosome comprising one or moremethicillin-resistant Staphylococcus aureus specific biomarkers, such aslisted in FIG. 57. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more methicillin-resistantStaphylococcus aureus specific biomarkers, such as listed in FIG. 57.The composition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for methicillin-resistant Staphylococcus aureus specificexosomes or exosomes comprising one or more methicillin-resistantStaphylococcus aureus specific biomarkers, such as listed in FIG. 57.

One or more methicillin-resistant Staphylococcus aureus specificbiomarkers, such as listed in FIG. 57, can also be detected by one ormore systems disclosed herein, for characterizing amethicillin-resistant Staphylococcus aureus. For example, a detectionsystem can comprise one or more probes to detect one or moremethicillin-resistant Staphylococcus aureus specific biomarkers, such aslisted in FIG. 57, of one or more exosomes of a biological sample.

Vulnerable Plaque

Vulnerable plaque specific biomarkers from exosomes can include one ormore (for example, 2, 3, 4, 5, 6, 7, 8, or more) overexpressed miRs,underexpressed miRs, mRNAs, genetic mutations, proteins, ligands,peptides, snoRNA, or any combination thereof, such as listed in FIG. 58,and can be used to create a vulnerable plaque specific exosomebio-signature. The protein, ligand, or peptide that can be assessed inan exosome can include, but is not limited to, IL-6, MMP-9, PAPP-A,D-dimer, fibrinogen, Lp-PLA2, SCD40L, Il-18, oxLDL, GPx-1, MCP-1, P1GF,or CRP, or any combination thereof.

Also provided herein is an isolated exosome comprising one or morevulnerable plaque specific biomarkers, such as listed in FIG. 58 and inFIG. 1 for vulnerable plaque. A composition comprising the isolatedexosome is also provided. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morevulnerable plaque specific biomarkers, such as listed in FIG. 58 and inFIG. 1 for vulnerable plaque. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for vulnerable plaque specificexosomes or exosomes comprising one or more vulnerable plaque specificbiomarkers, such as listed in FIG. 58 and in FIG. 1 for vulnerableplaque.

One or more vulnerable plaque specific biomarkers, such as listed inFIG. 58 and in FIG. 1 for vulnerable plaque, can also be detected by oneor more systems disclosed herein, for characterizing a vulnerableplaque. For example, a detection system can comprise one or more probesto detect one or more vulnerable plaque specific biomarkers, such aslisted in FIG. 58 and in FIG. 1 for vulnerable plaque, of one or moreexosomes of a biological sample.

Autoimmune Disease

Also provided herein is an isolated exosome comprising one or moreautoimmune disease specific biomarkers, such as listed in FIG. 1 forautoimmune disease. A composition comprising the isolated exosome isalso provided. Accordingly, in some embodiments, the compositioncomprises a population of exosomes comprising one or more autoimmunedisease specific biomarkers, such as listed in FIG. 1 for autoimmunedisease. The composition can comprise a substantially enrichedpopulation of exosomes, wherein the population of exosomes issubstantially homogeneous for autoimmune disease specific exosomes orexosomes comprising one or more autoimmune disease specific biomarkers,such as listed in FIG. 1 for autoimmune disease.

One or more autoimmune disease specific biomarkers, such as listed inFIG. 1 for autoimmune disease, can also be detected by one or moresystems disclosed herein, for characterizing a autoimmune disease. Forexample, a detection system can comprise one or more probes to detectone or more autoimmune disease specific biomarkers, such as listed inFIG. 1 for autoimmune disease, of one or more exosomes of a biologicalsample.

Tuberculosis (TB)

Also provided herein is an isolated exosome comprising one or more TBdisease specific biomarkers, such as listed in FIG. 1 for TB disease. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more TB disease specific biomarkers, suchas listed in FIG. 1 for TB disease. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for TB disease specific exosomesor exosomes comprising one or more TB disease specific biomarkers, suchas listed in FIG. 1 for TB disease.

One or more TB disease specific biomarkers, such as listed in FIG. 1 forTB disease, can also be detected by one or more systems disclosedherein, for characterizing a TB disease. For example, a detection systemcan comprise one or more probes to detect one or more TB diseasespecific biomarkers, such as listed in FIG. 1 for TB disease, of one ormore exosomes of a biological sample.

HIV

Also provided herein is an isolated exosome comprising one or more HIVdisease specific biomarkers, such as listed in FIG. 1 for HIV disease. Acomposition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more HIV disease specific biomarkers, suchas listed in FIG. 1 for HIV disease. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for HIV disease specific exosomesor exosomes comprising one or more HIV disease specific biomarkers, suchas listed in FIG. 1 for HIV disease.

One or more HIV disease specific biomarkers, such as listed in FIG. 1for HIV disease, can also be detected by one or more systems disclosedherein, for characterizing a HIV disease. For example, a detectionsystem can comprise one or more probes to detect one or more HIV diseasespecific biomarkers, such as listed in FIG. 1 for HIV disease, of one ormore exosomes of a biological sample.

The one or more biomarker can also be a miRNA, such as an upregulated oroverexpressed miRNA. The upregulated miRNA can be miR-29a, miR-29b,miR-149, miR-378 or miR-324-5p. One or more biomarkers can also be usedto characterize HIV-1 latency, such as by assessing one or more miRNAs.The miRNA can be miR-28, miR-125b, miR-150, miR-223 and miR-382, andupregulated.

Asthma

Also provided herein is an isolated exosome comprising one or moreasthma disease specific biomarkers, such as listed in FIG. 1 for asthmadisease. A composition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more asthma disease specific biomarkers,such as listed in FIG. 1 for asthma disease. The composition cancomprise a substantially enriched population of exosomes, wherein thepopulation of exosomes is substantially homogeneous for asthma diseasespecific exosomes or exosomes comprising one or more asthma diseasespecific biomarkers, such as listed in FIG. 1 for asthma disease.

One or more asthma disease specific biomarkers, such as listed in FIG. 1for asthma disease, can also be detected by one or more systemsdisclosed herein, for characterizing a asthma disease. For example, adetection system can comprise one or more probes to detect one or moreasthma disease specific biomarkers, such as listed in FIG. 1 for asthmadisease, of one or more exosomes of a biological sample.

Lupus

Also provided herein is an isolated exosome comprising one or more lupusdisease specific biomarkers, such as listed in FIG. 1 for lupus disease.A composition comprising the isolated exosome is also provided.Accordingly, in some embodiments, the composition comprises a populationof exosomes comprising one or more lupus disease specific biomarkers,such as listed in FIG. 1 for lupus disease. The composition can comprisea substantially enriched population of exosomes, wherein the populationof exosomes is substantially homogeneous for lupus disease specificexosomes or exosomes comprising one or more lupus disease specificbiomarkers, such as listed in FIG. 1 for lupus disease.

One or more lupus disease specific biomarkers, such as listed in FIG. 1for lupus disease, can also be detected by one or more systems disclosedherein, for characterizing a lupus disease. For example, a detectionsystem can comprise one or more probes to detect one or more lupusdisease specific biomarkers, such as listed in FIG. 1 for lupus disease,of one or more exosomes of a biological sample.

Influenza

Also provided herein is an isolated exosome comprising one or moreinfluenza disease specific biomarkers, such as listed in FIG. 1 forinfluenza disease. A composition comprising the isolated exosome is alsoprovided. Accordingly, in some embodiments, the composition comprises apopulation of exosomes comprising one or more influenza disease specificbiomarkers, such as listed in FIG. 1 for influenza disease. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for influenza disease specific exosomes or exosomescomprising one or more influenza disease specific biomarkers, such aslisted in FIG. 1 for influenza disease.

One or more influenza disease specific biomarkers, such as listed inFIG. 1 for influenza disease, can also be detected by one or moresystems disclosed herein, for characterizing a influenza disease. Forexample, a detection system can comprise one or more probes to detectone or more influenza disease specific biomarkers, such as listed inFIG. 1 for influenza disease, of one or more exosomes of a biologicalsample.

Thyroid Cancer

Also provided herein is an isolated exosome comprising one or morethyroid cancer specific biomarkers, such as AKAP-BRAF, CCDC6-RET,ERC1-RETM, GOLGA5-RET, HOOK3-RET, HRH4-RET, KTN1-RET, NCOA4-RET,PCM1-RET, PRKARA1A-RET, RFG-RET, RFG9-RET, Ria-RET, TGF-NTRK1,TPM3-NTRK1, TPM3-TPR, TPR-MET, TPR-NTRK1, TRIM24-RET, TRIM27-RET orTRIM33-RET, characteristic of papillary thyroid carcinoma; orPAX8-PPARy, characteristic of follicular thyroid cancer. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more thyroid cancer specific biomarkers, such aslisted in FIG. 1 for thyroid cancer. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for thyroid cancer specificexosomes or exosomes comprising one or more thyroid cancer specificbiomarkers, such as listed in FIG. 1 for thyroid cancer.

One or more thyroid cancer specific biomarkers, such as listed in FIG. 1for thyroid cancer, can also be detected by one or more systemsdisclosed herein, for characterizing a thyroid cancer. For example, adetection system can comprise one or more probes to detect one or morethyroid cancer specific biomarkers, such as listed in FIG. 1 for thyroidcancer, of one or more exosomes of a biological sample.

Gene Fusions

The one or more biomarkers assessed of an exosome can be a gene fusion,such as one or more listed in FIG. 59. A fusion gene is a hybrid genecreated by the juxtaposition of two previously separate genes. This canoccur by chromosomal translocation or inversion, deletion or viatrans-splicing. The resulting fusion gene can cause abnormal temporaland spatial expression of genes, such as leading to abnormal expressionof cell growth factors, angiogenesis factors, tumor promoters or otherfactors contributing to the neoplastic transformation of the cell andthe creation of a tumor. Such fusion genes can be oncogenic due to thejuxtaposition of: 1) a strong promoter region of one gene next to thecoding region of a cell growth factor, tumor promoter or other genepromoting oncogenesis leading to elevated gene expression, or 2) due tothe fusion of coding regions of two different genes, giving rise to achimeric gene and thus a chimeric protein with abnormal activity.

An example of a fusion gene is BCR-ABL, a characteristic molecularaberration in ˜90% of chronic myelogenous leukemia (CML) and in a subsetof acute leukemias (Kurzrock et al., Annals of Internal Medicine 2003;138(10): 819-830). The BCR-ABL results from a translocation betweenchromosomes 9 and 22. The translocation brings together the 5′ region ofthe BCR gene and the 3′ region of ABL1, generating a chimeric BCR-ABL1gene, which encodes a protein with constitutively active tyrosine kinaseactivity (Mittleman et al., Nature Reviews Cancer 2007; 7(4):233-245).The aberrant tyrosine kinase activity leads to de-regulated cellsignaling, cell growth and cell survival, apoptosis resistance andgrowth factor independence, all of which contribute to thepathophysiology of leukemia (Kurzrock et al., Annals of InternalMedicine 2003; 138(10):819-830).

Another fusion gene is IGH-MYC, a defining feature of ˜80% of Burkitt'slymphoma (Ferry et al. Oncologist 2006; 11(4):375-83). The causal eventfor this is a translocation between chromosomes 8 and 14, bringing thec-Myc oncogene adjacent to the strong promoter of the immunoglobin heavychain gene, causing c-myc overexpression (Mittleman et al., NatureReviews Cancer 2007; 7(4):233-245). The c-myc rearrangement is a pivotalevent in lymphomagenesis as it results in a perpetually proliferativestate. It has wide ranging effects on progression through the cellcycle, cellular differentiation, apoptosis, and cell adhesion (Ferry etal. Oncologist 2006; 11(4):375-83).

A number of recurrent fusion genes have been catalogued in the Mittlemandatabase (http://cgap.nci.nih.gov/Chromosomes/Mitelman) and can beassess in an exosome and used to characterize a phenotype. The genefusion can be used to characterize a hematological malignancy orepithelial tumor. For example, TMPRSS2-ERG, TMPRSS2-ETV and SLC45A3-ELK4fusions can be detected and used to characterize prostate cancer; andETV6-NTRK3 and ODZ4-NRG 1 for breast cancer.

Furthermore, assessing the presence or absence, or expression level of afusion gene can be used to diagnosis a phenotype such as a cancer aswell as a monitoring a therapeutic response to selecting a treatment.For example, the presence of the BCR-ABL fusion gene is a characteristicnot only for the diagnosis of CML, but is also the target of theNovartis drug Imatinib mesylate (Gleevec), a receptor tyrosine kinaseinhibitor, for the treatment of CML. Imatinib treatment has led tomolecular responses (disappearance of BCR-ABL+blood cells) and improvedprogression-free survival in BCR-ABL+CML patients (Kantarjian et al.,Clinical Cancer Research 2007; 13(4):1089-1097).

Assessing an exosome for the presence, absence, or expression level of agene fusion can be of a heterogeneous population of exosomes.Alternatively, the exosome can be derived from a specific cell type,such as cell-or-origin specific exosomes, as described above.

Breast Cancer

To characterize a breast cancer, an exosome can be assessed for one ormore breast cancer specific fusions, including, but not limited to,ETV6-NTRK3. The exosome can be derived from breast cancer cells.

Lung Cancer

To characterize a lung cancer, an exosome can be assessed for one ormore lung cancer specific fusions, including, but not limited to,RLF-MYCL1, TGF-ALK, or CD74-ROS1. The exosome can be derived from lungcancer cells.

Prostate Cancer

To characterize a prostate cancer, an exosome can be assessed for one ormore prostate cancer specific fusions, including, but not limited to,ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG,TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5or KLK2-ETV4. The exosome can be derived from prostate cancer cells.

Brain Cancer

To characterize a brain cancer, an exosome can be assessed for one ormore brain cancer specific fusions, including, but not limited to,GOPC-ROS1. The exosome can be derived from brain cancer cells.

Head and Neck Cancer

To characterize ahead and neck cancer, an exosome can be assessed forone or more head and neck cancer specific fusions, including, but notlimited to, CHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB,LIFR-PLAG1, or TCEA1-PLAG1. The exosome can be derived from head andneck cancer cells.

Renal Cell Carcinoma (RCC)

To characterize a RCC, an exosome can be assessed for one or more RCCspecific fusions, including, but not limited to, ALPHA-TFEB, NONO-TFE3,PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3, or MALAT1-TFEB. The exosome can bederived from RCC cells.

Thyroid Cancer

To characterize a thyroid cancer, an exosome can be assessed for one ormore thyroid cancer specific fusions, including, but not limited to,AKAP9-BRAF, CCDC6-RET, ERC1-RETM, GOLGA5-RET, HOOK3-RET, HRH4-RET,KTN1-RET, NCOA4-RET, PCM1-RET, PRKARA1A-RET, RFG-RET, RFG9-RET, R1a—RET,TGF-NTRK1, TPM3-NTRK1, TPM3-TPR, TPR-MET, TPR-NTRK1, TRIM24-RET,TRIM27-RET or TRIM33-RET, characteristic of papillary thyroid carcinoma;or PAX8-PPARy, characteristic of follicular thyroid cancer. The exosomecan be derived from thyroid cancer cells.

Blood Cancers

To characterize a blood cancer, an exosome can be assessed for one ormore blood cancer specific fusions, including, but not limited to,TTL-ETV6, CDK6-MLL, CDK6-TLX3, ETV6-FLT3, ETV6-RUNX1, ETV6-TTL,MLL-AFF1, MLL-AFF3, MLL-AFF4, MLL-GAS7, TCBA1-ETV6, TCF3-PBX1 orTCF3-TFPT, characteristic of acute lymphocytic leukemia (ALL);BCL11B-TLX3, IL2-TNFRFS17, NUP214-ABL1, NUP98-CCDC28A, TALI-STIL, orETV6-ABL2, characteristic of T-cell acute lymphocytic leukemia (T-ALL);ATIC-ALK, KIAA1618-ALK, MSN-ALK, MYH9-ALK, NPM1-ALK, TGF-ALK orTPM3-ALK, characteristic of anaplastic large cell lymphoma (ALCL);BCR-ABL1, BCR-JAK2, ETV6-EVI1, ETV6-MN1 or ETV6-TCBA1, characteristic ofchronic myelogenous leukemia (CML); CBFB-MYH11, CHIC2-ETV6, ETV6-ABL1,ETV6-ABL2, ETV6-ARNT, ETV6-CDX2, ETV6-HLXB9, ETV6-PER1, MEF2D-DAZAP1,AML-AFF1, MLL-ARHGAP26, MLL-ARHGEF12, MLL-CASC5, MLL-CBL, MLL-CREBBP,MLL-DAB21P, MLL-ELL, MLL-EP300, MLL-EPS15, MLL-FNBP1, MLL-FOXO3A,MLL-GMPS, MLL-GPHN, MLL-MLLT1, MLL-MLLT11, MLL-MLLT3, MLL-MLLT6,MLL-MYO1F, MLL-PICALM, MLL-SEPT2, MLL-SEPT6, MLL-SORBS2, MYST3-SORBS2,MYST-CREBBP, NPM1-MLF1, NUP98-HOXA13, PRDM16-EVI1, RABEP1-PDGFRB,RUNX1-EVI1, RUNX1-MDS1, RUNX1-RPL22, RUNX1-RUNX1T1, RUNX1-SH3D19,RUNX1-USP42, RUNX1-YTHDF2, RUNX1-ZNF687, or TAF15-ZNF-384,characteristic of AML; CCND1-FSTL3, characteristic of chroniclymphocytic leukemia (CLL); BCL3-MYC, MYC-BTG1, BCL7A-MYC,BRWD3-ARHGAP20 or BTG1-MYC, characteristic of B-cell chronic lymphocyticleukemia (B-CLL); CITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6,TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK, characteristic of diffuse largeB-cell lymphomas (DLBCL); FLIP1-PDGFRA, FLT3-ETV6, KIAA1509-PDGFRA,PDE4DIP-PDGFRB, NIN-PDGFRB, TP53BP1-PDGFRB, or TPM3-PDGFRB,characteristic of hyper eosinophilia/chronic eosinophilia; IGH-MYC orLCP1-BCL6, characteristic of Burkitt's lymphoma. The exosome can bederived from blood cancer cells.

Also provided herein is an isolated exosome comprising one or more genefusions as disclosed herein, such as listed in FIG. 59. A compositioncomprising the isolated exosome is also provided. Accordingly, in someembodiments, the composition comprises a population of exosomescomprising one or more gene fusions, such as listed in FIG. 59. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more gene fusions, such aslisted in FIG. 59.

Also provided herein is a detection system for detecting one or moregene fusions, such as gene fusions listed in FIG. 59. For example, adetection system can comprise one or more probes to detect one or moregene fusions listed in FIG. 59. Detection of the one or more genefusions can be used to characterize a cancer.

Gene-Associated mRNA Biomarkers

The one or more biomarkers assessed can also include one or more genesselected from the group consisting of PFKFB3, RHAMM (HMMR), cDNAFLJ42103, ASPM, CENPF, NCAPG, Androgen Receptor, EGFR, HSP90, SPARC,DNMT3B, GART, MGMT, SSTR3, and TOP2B. The microRNA that interacts withthe one or more genes can also be a biomarker (see for example, FIG.60). Furthermore, the one or more biomarkers can be used to characterizeprostate cancer.

Also provided herein is an isolated exosome comprising one or more oneor more biomarkers consisting of PFKFB3, RHAMM (HMMR), cDNA FLJ42103,ASPM, CENPF, NCAPG, Androgen Receptor, EGFR, HSP90, SPARC, DNMT3B, GART,MGMT, SSTR3, and TOP2B; or the microRNA that interacts with the one ormore genes (see for example, FIG. 60). Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of PFKFB3, RHAMM (HMMR), cDNA FLJ42103, ASPM,CENPF, NCAPG, Androgen Receptor, EGFR, HSP90, SPARC, DNMT3B, GART, MGMT,SSTR3, and TOP2B; or the microRNA that interacts with the one or moregenes, such as listed in FIG. 60. The composition can comprise asubstantially enriched population of exosomes, wherein the population ofexosomes is substantially homogeneous for exosomes comprising one ormore biomarkers consisting of PFKFB3, RHAMM (HMMR), cDNA FLJ42103, ASPM,CENPF, NCAPG, Androgen Receptor, EGFR, HSP90, SPARC, DNMT3B, GART, MGMT,SSTR3, and TOP2B; or the microRNA that interacts with the one or moregenes, such as listed in FIG. 60.

One or more prostate cancer specific biomarkers, such as listed in FIG.60 can also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more prostate cancer specific biomarkers, such as listed in FIG.60, of one or more exosomes of a biological sample.

The miRNA that interacts with PFKFB3 can be miR-513a-3p, miR-128,miR-488, miR-539, miR-658, miR-524-5p, miR-1258, miR-150, miR-216b,miR-377, miR-135a, miR-26a, miR-548a-5p, miR-26b, miR-520d-5p, miR-224,miR-1297, miR-1197, miR-182, miR-452, miR-509-3-5p, miR-548m, miR-625,miR-509-5p, miR-1266, miR-135b, miR-190b, miR-496, miR-616, miR-621,miR-650, miR-105, miR-19a, miR-346, miR-620, miR-637, miR-651, miR-1283,miR-590-3p, miR-942, miR-1185, miR-577, miR-602, miR-1305, miR-220c,miR-1270, miR-1282, miR-432, miR-491-5p, miR-548n, miR-765, miR-768-3por miR-924, and can be used as a biomarker.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with PFKFB3. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with PFKFB3. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith PFKFB3. Furthermore, the one or more miRNA that interacts withPFKFB3 can also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with PFKFB3 of one or moreexosomes of a biological sample.

The miRNA that interacts with RHAMM can be miR-936, miR-656, miR-105,miR-361-5p, miR-194, miR-374a, miR-590-3p, miR-186, miR-769-5p,miR-892a, miR-380, miR-875-3p, miR-208a, miR-208b, miR-586, miR-125a-3p,miR-630, miR-374b, miR-411, miR-629, miR-1286, miR-1185, miR-16,miR-200b, miR-671-5p, miR-95, miR-421, miR-496, miR-633, miR-1243,miR-127-5p, miR-143, miR-15b, miR-200c, miR-24 or miR-34c-3p.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with RHAMM. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with RHAMM. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith RHAMM. Furthermore, the one or more miRNA that interacts with RHAMMcan also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with RHAMM of one or moreexosomes of a biological sample.

The miRNA that interacts with CENPF can be miR-30c, miR-30b, miR-190,miR-508-3p, miR-384, miR-512-5p, miR-548p, miR-297, miR-520f, miR-376a,miR-1184, miR-577, miR-708, miR-205, miR-376b, miR-520g, miR-520h,miR-519d, miR-596, miR-768-3p, miR-340, miR-620, miR-539, miR-567,miR-671-5p, miR-1183, miR-129-3p, miR-636, miR-106a, miR-1301, miR-17,miR-20a, miR-570, miR-656, miR-1263, miR-1324, miR-142-5p, miR-28-5p,miR-302b, miR-452, miR-520d-3p, miR-548o, miR-892b, miR-302d,miR-875-3p, miR-106b, miR-1266, miR-1323, miR-20b, miR-221, miR-520e,miR-664, miR-920, miR-922, miR-93, miR-1228, miR-1271, miR-30e,miR-483-3p, miR-509-3-5p, miR-515-3p, miR-519e, miR-520b, miR-520c-3p ormiR-582-3p.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with CENPF. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with CENPF. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith CENPF. Furthermore, the one or more miRNA that interacts with CENPFcan also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with CENPF of one or moreexosomes of a biological sample.

The miRNA that interacts with NCAPG can be miR-876-5p, miR-1260,miR-1246, miR-548c-3p, miR-1224-3p, miR-619, miR-605, miR-490-5p,miR-186, miR-448, miR-129-5p, miR-188-3p, miR-516b, miR-342-3p,miR-1270, miR-548k, miR-654-3p, miR-1290, miR-656, miR-34b, miR-520g,miR-1231, miR-1289, miR-1229, miR-23a, miR-23b, miR-616 or miR-620.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with NCAPG. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with NCAPG. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith NCAPG. Furthermore, the one or more miRNA that interacts with NCAPGcan also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with NCAPG of one or moreexosomes of a biological sample.

The miRNA that interacts with Androgen Receptor can be miR-124a,miR-130a, miR-130b, miR-143, miR-149, miR-194, miR-29b, miR-29c,miR-301, miR-30a-5p, miR-30d, miR-30e-5p, miR-337, miR-342, miR-368,miR-488, miR-493-5p, miR-506, miR-512-5p, miR-644, miR-768-5p ormiR-801.

The miRNA that interacts with EGFR can be miR-105, miR-128a, miR-128b,miR-140, miR-141, miR-146a, miR-146b, miR-27a, miR-27b, miR-302a,miR-302d, miR-370, miR-548c, miR-574, miR-587 or miR-7.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with AR. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with AR. The compositioncan comprise a substantially enriched population of exosomes, whereinthe population of exosomes is substantially homogeneous for exosomescomprising one or more miRNA that interacts with AR. Furthermore, theone or more miRNA that interacts with AR can also be detected by one ormore systems disclosed herein. For example, a detection system cancomprise one or more probes to detect one or more one or more miRNA thatinteracts with AR of one or more exosomes of a biological sample.

The miRNA that interacts with HSP90 can be miR-1, miR-513a-3p,miR-548d-3p, miR-642, miR-206, miR-450b-3p, miR-152, miR-148a, miR-148b,miR-188-3p, miR-23a, miR-23b, miR-578, miR-653, miR-1206, miR-192,miR-215, miR-181b, miR-181d, miR-223, miR-613, miR-769-3p, miR-99a,miR-100, miR-454, miR-548n, miR-640, miR-99b, miR-150, miR-181a,miR-181c, miR-522, miR-624, miR-130a, miR-130b, miR-146, miR-148a,miR-148b, miR-152, miR-181a, miR-181b, miR-181c, miR-204, miR-206,miR-211, miR-212, miR-215, miR-223, miR-23a, miR-23b, miR-301, miR-31,miR-325, miR-363, miR-566, miR-9 or miR-99b.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with HSP90. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with HSP90. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith HSP90. Furthermore, the one or more miRNA that interacts with HSP90can also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with HSP90 of one or moreexosomes of a biological sample.

The miRNA that interacts with SPARC can be miR-768-5p, miR-203,miR-196a, miR-569, miR-187, miR-641, miR-1275, miR-432, miR-622,miR-296-3p, miR-646, miR-196b, miR-499-5p, miR-590-5p, miR-495, miR-625,miR-1244, miR-512-5p, miR-1206, miR-1303, miR-186, miR-302d, miR-494,miR-562, miR-573, miR-10a, miR-203, miR-204, miR-211, miR-29, miR-29b,miR-29c, miR-339, miR-433, miR-452, miR-515-5p, miR-517a, miR-517b,miR-517c, miR-592 or miR-96.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with SPARC. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with SPARC. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith SPARC. Furthermore, the one or more miRNA that interacts with SPARCcan also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with SPARC of one or moreexosomes of a biological sample.

The miRNA that interacts with DNMT3B can be miR-618, miR-1253, miR-765,miR-561, miR-330-5p, miR-326, miR-188, miR-203, miR-221, miR-222,miR-26a, miR-26b, miR-29a, miR-29b, miR-29c, miR-370, miR-379, miR-429,miR-519e, miR-598, miR-618 or miR-635.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with DNMT3B. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with DNMT3B. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith DNMT3B. Furthermore, the one or more miRNA that interacts withDNMT3B can also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with DNMT3B of one or moreexosomes of a biological sample.

The miRNA that interacts with GART can be miR-101, miR-141, miR-144,miR-182, miR-189, miR-199a, miR-199b, miR-200a, miR-200b, miR-202,miR-203, miR-223, miR-329, miR-383, miR-429, miR-433, miR-485-5p,miR-493-5p, miR-499, miR-519a, miR-519b, miR-519c, miR-569, miR-591,miR-607, miR-627, miR-635, miR-636 or miR-659.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with GART. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with GART. The compositioncan comprise a substantially enriched population of exosomes, whereinthe population of exosomes is substantially homogeneous for exosomescomprising one or more miRNA that interacts with GART. Furthermore, theone or more miRNA that interacts with GART can also be detected by oneor more systems disclosed herein. For example, a detection system cancomprise one or more probes to detect one or more one or more miRNA thatinteracts with GART of one or more exosomes of a biological sample.

The miRNA that interacts with MGMT can be miR-122a, miR-142-3p,miR-17-3p, miR-181a, miR-181b, miR-181c, miR-181d, miR-199b, miR-200a,miR-217, miR-302b, miR-32, miR-324-3p, miR-34a, miR-371, miR-425-5p,miR-496, miR-514, miR-515-3p, miR-516-3p, miR-574, miR-597, miR-603,miR-653, miR-655, miR-92, miR-92b or miR-99a.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with MGMT. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with MGMT. The compositioncan comprise a substantially enriched population of exosomes, whereinthe population of exosomes is substantially homogeneous for exosomescomprising one or more miRNA that interacts with MGMT. Furthermore, theone or more miRNA that interacts with MGMT can also be detected by oneor more systems disclosed herein. For example, a detection system cancomprise one or more probes to detect one or more one or more miRNA thatinteracts with MGMT of one or more exosomes of a biological sample.

The miRNA that interacts with SSTR3 can be miR-125a, miR-125b, miR-133a,miR-133b, miR-136, miR-150, miR-21, miR-380-5p, miR-504, miR-550,miR-671, miR-766 or miR-767-3p.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with SSTR3. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with SSTR3. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith SSTR3. Furthermore, the one or more miRNA that interacts with SSTR3can also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with SSTR3 of one or moreexosomes of a biological sample.

The miRNA that interacts with TOP2B can be miR-548f, miR-548a-3p,miR-548g, miR-513a-3p, miR-548c-3p, miR-101, miR-653, miR-548d-3p,miR-575, miR-297, miR-576-3p, miR-548b-3p, miR-624, miR-548n, miR-758,miR-1253, miR-1324, miR-23b, miR-320a, miR-320b, miR-1183, miR-1244,miR-23a, miR-451, miR-568, miR-1276, miR-548e, miR-590-3p, miR-1,miR-101, miR-126, miR-129, miR-136, miR-140, miR-141, miR-144, miR-147,miR-149, miR-18, miR-181b, miR-181c, miR-182, miR-184, miR-186, miR-189,miR-191, miR-19a, miR-19b, miR-200a, miR-206, miR-210, miR-218, miR-223,miR-23a, miR-23b, miR-24, miR-27a, miR-302, miR-30a, miR-31, miR-320,miR-323, miR-362, miR-374, miR-383, miR-409-3p, miR-451, miR-489,miR-493-3p, miR-514, miR-542-3p, miR-544, miR-548a, miR-548b, miR-548c,miR-548d, miR-559, miR-568, miR-575, miR-579, miR-585, miR-591, miR-598,miR-613, miR-649, miR-651, miR-758, miR-768-3p or miR-9.

Also provided herein is an isolated exosome comprising one or more oneor more miRNA that interacts with TOP2B. Also provided is a compositioncomprising the isolated exosome. Accordingly, in some embodiments, thecomposition comprises a population of exosomes comprising one or morebiomarkers consisting of miRNA that interacts with TOP2B. Thecomposition can comprise a substantially enriched population ofexosomes, wherein the population of exosomes is substantiallyhomogeneous for exosomes comprising one or more miRNA that interactswith TOP2B. Furthermore, the one or more miRNA that interacts with TOP2Bcan also be detected by one or more systems disclosed herein. Forexample, a detection system can comprise one or more probes to detectone or more one or more miRNA that interacts with TOP2B of one or moreexosomes of a biological sample.

Bio-Signatures: Biomarker Detection

Bio-signatures can be detected qualitatively or quantitatively. Exosomelevels may be characterized as described above. Analysis of exosomes cancomprise detecting the level of exosomes in combination with determiningthe biomarkers of the exosomes. Determining the level or amount ofexosome can be performed in conjunction with determining the biomarkersof the exosome. Alternatively, determining the amount of exosome may beperformed prior to or subsequent to determining the biomarkers of theexosomes. Methods for analyzing biomarkers of tissues or cells can beused to analyze the biomarkers associated with or contained in exosomes.

For example, biomarkers can be detected by microarray analysis, PCR(including PCR-based methods such as RT-PCR, qPCR and the like),hybridization with allele-specific probes, enzymatic mutation detection,ligation chain reaction (LCR), oligonucleotide ligation assay (OLA),flow-cytometric heteroduplex analysis, chemical cleavage of mismatches,mass spectrometry, nucleic acid sequencing, single strand conformationpolymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE),temperature gradient gel electrophoresis (TGGE), restriction fragmentpolymorphisms, serial analysis of gene expression (SAGE), or anycombinations thereof. The biomarker, such as a nucleic acid, can beamplified prior to detection. Biomarkers can also be detected byimmunoblot, immunoprecipitation, ELISA, RIA, flow cytometry, or electronmicroscopy.

One method of detecting biomarkers can include purifying or isolating aheterogeneous exosome population from a biological sample, as describedabove, and performing a sandwich assay. An exosome in the population canbe captured with a primary antibody, such as an antibody bound to asubstrate, for example an array, well, or particle. The captured orbound exosome can be detected with a detection antibody. For example,the detection antibody can be for an antigen of the exosome. Thedetection antibody can be directly labeled and detected. Alternatively,an enzyme linked secondary antibody can react with the detectionantibody. A detection reagent or detection substrate is added and thereaction can be detected, such as described in PCT Publication No.WO2009092386. The primary antibody can be an anti-Rab 5b antibody andthe detection antibody anti-CD63 or anti-caveolin-1. Alternatively, thecapture antibody can be an antibody to CD9, PSCA, TNFR, CD63, B7H3,MFG-E8, EpCam, Rab, CD81, STEAP, PCSA, PSMA, or 5T4. The detectionantibody can be an antibody to CD63, CD9, CD81, B7H3, or EpCam.

In some embodiments, the capture agent binds or targets EpCam, and theone or more biomarkers detected on the exosome is CD9, CD63, or both CD9and CD63. In other embodiments, the capture agent targets PCSA, and theone or more biomarkers detected on the captured exosome is B7H3, PSMA,or both B7H3 and PSMA. In yet other embodiments, the capture agenttargets CD63 and the one or more biomarkers detected on the exosome isCD81, CD83, CD9, CD63, or any combination thereof. The different captureagent and biomarker combinations can be used to characterize aphenotype, such as prostate cancer or colon cancer. For example,capturing one or more exosomes can be performed with a capture agenttargeting EpCam and detection of CD9 and CD63; a capture agent targetingPCSA and detection of B7H3 and PSMA; or a capture agent of CD63 anddetection of CD81; can be used to characterize prostate cancer. Acapture agent targeting CD63 and detection of CD63, or a capture agenttargeting CD9 and detecting CD63, can be used to characterize coloncancer.

Other methods can include the use of a planar substrate such as an array(i.e., biochip or microarray), with immobilized molecules as captureagents, which can facilitate the detection of a particular bio-signatureof exosomes. The arrays can be provided as part of a kit for assayingexosomes. Molecules that identify the biomarkers described above andshown in FIG. 3-60, as well as antigens in FIG. 1 can be included in acustom array for detection and diagnosis of diseases includingpresymtomatic diseases. Arrays comprising biomolecules that specificallyidentify selected biomarkers can be used to develop a database ofinformation using data provided in the present specification. Additionalbiomolecules that identify bio-signatures which lead to improvedcross-validated error rates in multivariate prediction models (e.g.,logistic regression, discriminant analysis, or regression tree models)can be included in a custom array.

Customized array(s) provide an opportunity to study the biology of adisease, condition or syndrome and profile exosomes that are shed indefined physiological states. Standard p values of significance (0.05)can be chosen to exclude or include additional specific biomolecules onthe microarray that identify particular biomarkers.

A planar array can generally contain addressable locations (e.g., pads,addresses, or micro-locations) of biomolecules in an array format. Thesize of the array will depend on the composition and end use of thearray. Arrays containing from about 2 different molecules to manythousands can be made. Generally, the array can comprise from two to asmany as 100,000 or more molecules, depending on the end use of the arrayand the method of manufacture. A microarray can generally comprise atleast one biomolecule that identifies or captures a biomarker present ina bio-signature of specific cell-of-origin exosomes. In someembodiments, the compositions of the invention may not be in an arrayformat; that is, for some embodiments, compositions comprising a singlebiomolecule may be made as well. In addition, in some arrays, multiplesubstrates may be used, either of different or identical compositions.Thus, for example, large planar arrays may comprise a plurality ofsmaller substrates.

An array of the present invention encompasses any means for detecting abiomarker. For example, microarrays can be biochips that providehigh-density immobilized arrays of recognition molecules (e.g.,antibodies), where biomarker binding is monitored indirectly (e.g., viafluorescence). In addition, an array can be of a format that involvesthe capture of proteins by biochemical or intermolecular interaction,coupled with direct detection by mass spectrometry (MS).

Arrays and microarrays that can be used to detect the biomarkers of abio-signature of exosomes can be made according to the methods describedin U.S. Pat. Nos. 6,329,209; 6,365,418; 6,406,921; 6,475,808; and6,475,809, and U.S. patent application Ser. No. 10/884,269, each ofwhich is herein incorporated by reference in its entirety. New arrays,to detect specific selections of sets of biomarkers described herein canalso be made using the methods described in these patents. Furthermore,commercially available microarrays, such as for protein or nucleic aciddetection can also be used, such as from Affymetrix (Santa Clara,Calif.), Illumina (San Diego, Calif.), Agilent (Santa Clara, Calif.),Exiqon (Denmark), or Invitrogen (Carlsbad, Calif.).

In many embodiments, immobilized molecules, or molecules to beimmobilized, are proteins or peptides. One or more types of proteins maybe immobilized on a surface. In certain embodiments, the proteins areimmobilized using methods and materials that minimize the denaturing ofthe proteins, that minimize alterations in the activity of the proteins,or that minimize interactions between the protein and the surface onwhich they are immobilized.

Surfaces useful may be of any desired shape (form) and size.Non-limiting examples of surfaces include chips, continuous surfaces,curved surfaces, flexible surfaces, films, plates, sheets, tubes, or thelike. Surfaces can have areas ranging from approximately a square micronto approximately 500 cm². The area, length, and width of surfacesaccording to the present invention may be varied according to therequirements of the assay to be performed. Considerations may include,for example, ease of handling, limitations of the material(s) of whichthe surface is formed, requirements of detection systems, requirementsof deposition systems (e.g., arrayers), or the like.

In certain embodiments, it is desirable to employ a physical means forseparating groups or arrays of binding islands or immobilizedbiomolecules: such physical separation facilitates exposure of differentgroups or arrays to different solutions of interest. Therefore, incertain embodiments, arrays are situated within microwell plates havingany number of wells. In such embodiments, the bottoms of the wells mayserve as surfaces for the formation of arrays, or arrays may be formedon other surfaces and then placed into wells. In certain embodiments,such as where a surface without wells is used, binding islands may beformed or molecules may be immobilized on a surface and a gasket havingholes spatially arranged so that they correspond to the islands orbiomolecules may be placed on the surface. Such a gasket is preferablyliquid tight. A gasket may be placed on a surface at any time during theprocess of making the array and may be removed if separation of groupsor arrays is no longer necessary.

The immobilized molecules can bind to exosomes present in a biologicalsample overlying the immobilized molecules. Alternatively, theimmobilized molecules modify or are modified by molecules present inexosomes overlying the immobilized molecules.

Modifications or binding of molecules in solution or immobilized on anarray may be detected using detection techniques known in the art.Examples of such techniques include immunological techniques such ascompetitive binding assays and sandwich assays; fluorescence detectionusing instruments such as confocal scanners, confocal microscopes, orCCD-based systems and techniques such as fluorescence, fluorescencepolarization (FP), fluorescence resonant energy transfer (FRET), totalinternal reflection fluorescence (TIRF), fluorescence correlationspectroscopy (FCS); colorimetric/spectrometric techniques; surfaceplasmon resonance, by which changes in mass of materials adsorbed atsurfaces may be measured; techniques using radioisotopes, includingconventional radioisotope binding and scintillation proximity assays(SPA); mass spectroscopy, such as matrix-assisted laserdesorption/ionization mass spectroscopy (MALDI) and MALDI-time of flight(TOF) mass spectroscopy; ellipsometry, which is an optical method ofmeasuring thickness of protein films; quartz crystal microbalance (QCM),a very sensitive method for measuring mass of materials adsorbing tosurfaces; scanning probe microscopies, such as AFM and SEM; andtechniques such as electrochemical, impedance, acoustic, microwave, andIR/Raman detection. See, e.g., Mere L, et al., “Miniaturized FRET assaysand microfluidics: key components for ultra-high-throughput screening,”Drug Discovery Today 4(8):363-369 (1999), and references cited therein;Lakowicz J R, Principles of Fluorescence Spectroscopy, 2nd Edition,Plenum Press (1999), or Jain K K: Integrative Omics, Pharmacoproteomics,and Human Body Fluids. In: Thongboonkerd V, ed., ed. Proteomics of HumanBody Fluids: Principles, Methods and Applications. Volume 1: Totowa,N.J.: Humana Press, 2007, each of which is herein incorporated byreference in its entirety.

Microarray technology can be combined with mass spectroscopy (MS)analysis and other tools. Electrospray interface to a mass spectrometercan be integrated with a capillary in microfluidics devices. Forexample, one commercially available system contains eTag reporters thatare fluorescent labels with unique and well-defined electrophoreticmobilities; each label is coupled to biological or chemical probes viacleavable linkages. The distinct mobility address of each eTag reporterallows mixtures of these tags to be rapidly deconvoluted and quantitatedby capillary electrophoresis. This system allows concurrent geneexpression, protein expression, and protein function analyses from thesame sample Jain K K: Integrative Omics, Pharmacoproteomics, and HumanBody Fluids. In: Thongboonkerd V, ed., ed. Proteomics of Human BodyFluids Principles, Methods and Applications. Volume 1: Totowa, N.J.:Humana Press, 2007, which is herein incorporated by reference in itsentirety.

These biochips can include components for microfluidic or nanofluidicassays. Microfluidic devices can be used for isolating exosomes, such asdescribed herein, in combination with analyzing the exosomes, such asdetermining bio-signatures. Such systems miniaturize andcompartmentalize processes that allow for capturing of exosomes,detection of exosomal biomarkers, and other processes. The microfluidicdevices can utilize detection reagents in at least one aspect of thesystem, and such detection reagents may be used to detect one or morebiomarkers of exosomes. For example, the device can detect biomarkers onthe isolated exosomes or bound exosomes. One or more biomarkers of asample of isolated exosomes can be detected through the use of amicrofluidic device. For example, various probes, antibodies, proteins,or other binding agents can be used to detect a biomarker. The detectionagents may be immobilized in different compartments of the microfluidicdevice or be entered into a hybridization or detection reaction throughvarious channels of the device.

An exosome in a microfluidic device may be lysed and the contents, suchas proteins or nucleic acids, such as DNA or RNA (such as miRNA, mRNA)can be detected within a microfluidic device. The nucleic acid may beamplified prior to detection, or directly detected, within themicrofluidic device. Thus microfluidic systems can also be used formultiplexing detection of various biomarkers.

Novel nanofabrication techniques are opening up the possibilities forbiosensing applications that rely on fabrication of high-density,precision arrays, e.g., nucleotide-based chips and protein arraysotherwise know as heterogeneous nanoarrays. Nanofluidics allows afurther reduction in the quantity of fluid analyte in a microchip tonanoliter levels, and the chips used here are referred to as nanochips.(See, e.g., Unger M et al., Biotechniques 1999; 27(5):1008-14, KartalovE P et al., Biotechniques 2006; 40(1):85-90, each of which are hereinincorporated by reference in their entireties.) Commercially availablenanochips currently provide simple one step assays such as totalcholesterol, total protein or glucose assays that can be run bycombining sample and reagents, mixing and monitoring of the reaction.Gel-free analytical approaches based on liquid chromatography (LC) andnanoLC separations (Cutillas et al. Proteomics, 2005; 5:101-112 andCutillas et al., Mol Cell Proteomics 2005; 4:1038-1051, each of which isherein incorporated by reference in its entirety) can be used incombination with the nanochips.

Arrays suitable for identifying a disease, condition or a syndrome orphysiological status may be included in kits. Such kits may alsoinclude, as non-limiting examples, reagents useful for preparingmolecules for immobilization onto binding islands or areas of an array,reagents useful for detecting binding of exosomes or exosomal componentsto immobilized molecules, and instructions for use.

Further provided herein is a rapid detection device that facilitates thedetection of a particular bio-signature of exosomes in a biologicalsample. The device can integrate biological sample preparation withpolymerase chain reaction (PCR) on a chip. The device can facilitate thedetection of a particular bio-signature of exosomes in a biologicalsample, and an example is provided as described in Pipper et al.,Angewandte Chemie, 47(21), p. 3900-3904 (2008), which is hereinincorporated by reference in its entirety. The bio-signatures of theexosomes can be incorporated using micro-/nano-electrochemical system(MEMS/NEMS) sensors and oral fluid for diagnostic applications asdescribed in Li et al., Adv Dent Res 18(1): 3-5 (2005), which is hereinincorporated by reference in its entirety.

As an alternative to planar arrays, assays using particles, such as beadbased assays as described herein, can be used in combination with flowcytometry. Multiparametric assays or other high throughput detectionassays using bead coatings with cognate ligands and reporter moleculeswith specific activities consistent with high sensitivity automation canbe used. In bead based assay systems, the binding agents, such as anantibody for exosomes, can be immobilized on addressable microspheres.Each binding agent for each individual binding assay is coupled to adistinct type of microsphere (i.e., microbead) and the assay reactiontakes place on the surface of the microspheres, such as depicted in FIG.64A. A binding agent for an exosome, such as a capture antibody iscoupled to a bead. Dyed microspheres with discrete fluorescenceintensities are loaded separately with their appropriate binding agentor capture probes. The different bead sets carrying different bindingagents can be pooled as necessary to generate custom bead arrays. Beadarrays are then incubated with the sample in a single reaction vessel toperform the assay. Examples of microfluidic devices that may be used, oradapted for use with exosomes, include but are not limited to thosedescribed herein.

Product formation of the biomarker with their immobilized capturemolecules or binding agents can be detected with a fluorescence basedreporter system (see for example, FIG. 64A). The biomarker can either belabeled directly by a fluorophore or detected by a second fluorescentlylabeled capture biomolecule. The signal intensities derived fromcaptured biomarkers are measured in a flow cytometer. The flow cytometerfirst identifies each microsphere by its individual color code. Forexample, distinct beads can be dyed with discrete fluorescenceintensities such that each bead with a different intensity has adifferent binding agent. The beads can be labeled or dyed with at least2 different labels or dyes. In some embodiments, the beads are labeledwith at least 3, 4, 5, 6, 7, 8, 9, or 10 different labels. The beadswith more than one label or dye can also have various ratios andcombinations of the labels or dyes. The beads can be labeled or dyedexternally or may have intrinsic fluorescence or signaling labels.

The amount of captured biomarkers on each individual bead can bemeasured by the second color fluorescence specific for the bound target.This allows multiplexed quantitation of multiple targets from a singlesample within the same experiment. Sensitivity, reliability and accuracyare compared, or can be improved to standard microtiter ELISAprocedures. An advantage of bead-based systems is the individualcoupling of the capture biomolecule, or binding agent for an exosome, todistinct microspheres, which provides multiplexing. For example, asdepicted in FIG. 64B, a combination of 5 different biomarkers to bedetected (detected by antibodies to antigens such as CD63, CD9, CD81,B7H3, and EpCam) and 20 biomarkers for which to capture the exosome(using capture antibodies, such as antibodies to CD9, PSCA, TNFR, CD63,B7H3, MFG-E8, EpCam, Rab, CD81, STEAP, PCSA, PSMA, and 5T4) can resultin 100 combinations to be detected. Thus, captured exosomes can bedetected using detection agents, such as antibodies. The detectionagents can be labeled directly or indirectly, such as described above.

Multiplexing of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 50, 75 or 100 different biomarkers may beperformed. For example, an assay of a heterogeneous population ofexosomes can be performed with a plurality of particles that aredifferentially labeled. There can be at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75 or 100differentially labeled particles. The particles may be externallylabeled, such as with a tag, or they may be intrinsically labeled. Eachdifferentially labeled particle can be coupled to a capture agent, suchas a binding agent, for an exosome, resulting in capture of an exosome.Biomarkers of the captured exosomes can then be detected by a pluralityof binding agents. The binding agent can be directly labeled and thus,detected. Alternatively, the binding agent is labeled by a secondaryagent. For example, the binding agent may be an antibody for a biomarkeron the exosome. The binding agent is linked to biotin. A secondary agentcomprises streptavidin linked to a reporter and can be added to detectthe biomarker. In some embodiments, the captured exosomes are assayedfor at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 50, 75 or 100 different biomarkers. For example, as depictedin FIG. 70, multiple detectors, i.e. detection of multiple biomarkers ofa captured exosome, can increase the signal obtained, permittedincreased sensitivity, specificity or both, and the use of smalleramounts of samples.

ELISA based methods, so sandwich assay can also be used to detectbiomarkers on an exosome. A binding agent or capture agent can be boundto a well, for example an antibody to an exosomal antigen. Biomarkers onthe captured exosome can be detected based on the methods describedherein.

Peptide or protein biomakers can be analyzed by mass spectrometry orflow cytometry. Proteomic analysis of exosomes may also be carried outon exosomes by immunocytochemical staining, Western blotting,electrophoresis, chromatography or x-ray crystallography in accordancewith procedures well known in the art. In other embodiments, the proteinbio-signatures of exosomes may be analyzed using 2 D differential gelelectrophoresis as described in, Chromy et al. J Proteome Res, 2004;3:1120-1127, which is herein incorporated by reference in its entirety,or with liquid chromatography mass spectrometry as described in Zhang etal. Mol Cell Proteomics, 2005; 4:144-155, which is herein incorporatedby reference in its entirety. Exosomes may be subjected toactivity-based protein profiling described for example, in Berger etal., Am J Pharmacogenomics, 2004; 4:371-381, which is in incorporated byreference in its entirety. In other embodiments, exosomes may beprofiled using nanospray liquid chromatography-tandem mass spectrometryas described in Pisitkun et al., Proc Natl Acad Sci USA, 2004;101:13368-13373, which is herein incorporated by reference in itsentirety. In another embodiment, the exosomes may be profiled usingtandem mass spectrometry (MS) such as liquid chromatography/MS/MS(LC-MS/MS) using for example a LTQ and LTQ-FT ion trap massspectrometer. Protein identification can be determined and relativequantitation can be assessed by comparing spectral counts as describedin Smalley et al., J Proteome Res, 2008; 7:2088-2096, which is hereinincorporated by reference in its entirety.

Protein expression of exosomes can also be identified, such as followingthe isolation of cell-of-origin specific exosomes, such exosomes can beresuspended in buffer, centrifuged at 100×g for example, for 3 minutesusing a cytocentrifuge on adhesive slides in preparation forimmunocytochemical staining. The cytospins can be air-dried overnightand stored at—80° C. until staining. Slides can then be fixed andblocked with serum-free blocking reagent. The slides can then beincubated with a specific antibody to detect the expression of a proteinof interest. In some embodiments, the exosomes are not purified,isolated or concentrated prior to protein expression analysis.

Exosomes, such as isolated cell-of-origin specific exosomes can becharacterized by analysis of metabolite markers or metabolites, whichcan also form a bio-signature for exosomes. Various metabolite-orientedapproaches have been described such as metabolite target analyses,metabolite profiling, or metabolic fingerprinting, see for example,Denkert et al., Molecular Cancer 2008; 7: 4598-4617, Ellis et al.,Analyst 2006; 8: 875-885, Kuhn et al., Clinical Cancer Research 2007;24: 7401-7406, Fiehn O., Comp Funct Genomics 2001; 2:155-168, Fancy etal., Rapid Commun Mass Spectrom 20(15): 2271-80 (2006), Lindon et al.,Pharm Res, 23(6): 1075-88 (2006), Holmes et al., Anal Chem. 2007 Apr. 1;79(7):2629-40. Epub 2007 February 27. Erratum in: Anal Chem. 2008 Aug.1; 80(15):6142-3, Stanley et al., Anal Biochem. 2005 Aug. 15;343(2):195-202., Lehtimäki et al., J Biol. Chem. 2003 Nov. 14;278(46):45915-23, each of which is herein incorporated by reference inits entirety.

Peptides from exosomes can be analyzed by systems described in Jain K K:Integrative Omics, Pharmacoproteomics, and Human Body Fluids. In:Thongboonkerd V. ed., ed. Proteomics of Human Body Fluids: Principles,Methods and Applications. Volume 1: Totowa, N.J.: Humana Press, c2007.,2007, which is herein incorporated by reference in its entirety. Thissystem can generate sensitive molecular fingerprints of proteins presentin a body fluid as well as in exosomes. Commercial applications whichinclude the use of chromatography/mass spectroscopy and referencelibraries of all stable metabolites in the human body, for exampleParadigm Genetic's Human Metabolome Project, may be used to determinethe metabolite bio-signature of exosomes, such as isolatedcell-of-origin specific exosomes. Other methods for analyzing ametabolic profile can include methods and devices described in U.S. Pat.No. 6,683,455 (Metabometrix), U.S. Patent Application Publication Nos.20070003965 and 20070004044 (Biocrates Life Science), each of which isherein incorporated by reference in its entirety. Other proteomicprofiling techniques are described in Kennedy, Toxicol Lett 120:379-384(2001), Berven et al., Curr Pharm Biotechnol 7(3): 147-58 (2006),Conrads et al., Expert Rev Proteomics 2(5): 693-703, Decramer et al.,World J Urol 25(5): 457-65 (2007), Decramer et al., Mol Cell Proteomics7(10): 1850-62 (2008), Decramer et al., Contrib Nephrol, 160: 127-41(2008), Diamandis, J Proteome Res 5(9): 2079-82 (2006), Immler et al.,Proteomics 6(10): 2947-58 (2006), Khan et al., J Proteome Res 5(10):2824-38 (2006), Kumar et al., Biomarkers 11(5): 385-405 (2006), Noble etal., Breast Cancer Res Treat 104(2): 191-6 (2007), Omenn, Dis Markers20(3): 131-4 (2004), Powell et al., Expert Rev Proteomics 3(1): 63-74(2006), Rai et al., Arch Pathol Lab Med, 126(12): 1518-26 (2002),Ramstrom et al., Proteomics, 3(2): 184-90 (2003), Tammen et al., BreastCancer Res Treat, 79(1): 83-93 (2003), Theodorescu et al., Lancet Oncol,7(3): 230-40 (2006), or Zurbig et al., Electrophoresis, 27(11): 2111-25(2006).

For analysis of mRNAs, miRNAs or other small RNAs, the total RNA can befirst isolated from exosomes using any other known methods for isolatingnucleic acids such as methods described in U.S. Patent ApplicationPublication No. 2008132694, which is herein incorporated by reference inits entirety. These include, but are not limited to, kits for performingmembrane based RNA purification, which are commercially available.Generally, kits are available for the small-scale (30 mg or less)preparation of RNA from cells and tissues, for the medium scale (250 mgtissue) preparation of RNA from cells and tissues, and for the largescale (1 g maximum) preparation of RNA from cells and tissues. Othercommercially available kits for effective isolation of smallRNA-containing total RNA are available.

Alternatively, RNA can be isolated using the method described in U.S.Pat. No. 7,267,950, which is herein incorporated by reference in itsentirety. U.S. Pat. No. 7,267,950 describes a method of extracting RNAfrom biological systems (cells, cell fragments, organelles, tissues,organs, or organisms) in which a solution containing RNA is contactedwith a substrate to which RNA can bind and RNA is withdrawn from thesubstrate by applying negative pressure. Alternatively, RNA may beisolated using the method described in U.S. Patent Application No.20050059024, which is herein incorporated by reference in its entirety,which describes the isolation of small RNA molecules. Other methods aredescribed in U.S. Patent Application No. 20050208510, 20050277121,20070238118, each of which is incorporated by reference in its entirety.

In one embodiment, mRNA expression analysis can be carried out on mRNAsfrom exosomes isolated from a sample. In some embodiments, the exosomesare cell-of-origin specific exosomes. Expression patterns generated fromthese exosomes can be indicative of a given disease state, diseasestage, therapy related signature, or physiological condition. Once thetotal RNA has been isolated, cDNA can be synthesized and either qRT-PCRassays (e.g. Applied Biosystem's Taqman® assays) for specific mRNAtargets can be performed according to manufacturer's protocol, or anexpression microarray can be performed to look at highly multiplexedsets of expression markers in one experiment. Methods for establishinggene expression profiles include determining the amount of RNA that isproduced by a gene that can code for a protein or peptide. This isaccomplished by quantitative reverse transcriptase PCR (qRT-PCR),competitive RT-PCR, real time RT-PCR, differential display RT-PCR,Northern Blot analysis or other related tests. While it is possible toconduct these techniques using individual PCR reactions, it is alsopossible to amplify complementary DNA (cDNA) or complementary RNA (cRNA)produced from mRNA and analyze it via microarray.

The level of a miRNA product in a sample can be measured using anytechnique that is suitable for detecting mRNA expression levels in abiological sample, including but not limited to Northern blot analysis,RT-PCR, qRT-PCR, in situ hybridization or microarray analysis. Forexample, using gene specific primers and target cDNA, qRT-PCR enablessensitive and quantitative miRNA measurements of either a small numberof target miRNAs (via singleplex and multiplex analysis) or the platformcan be adopted to conduct high throughput measurements using 96-well or384-well plate formats. See for example, Ross J S et al, Oncologist.2008 May; 13(5):477-93, which is herein incorporated by reference in itsentirety. A number of different array configurations and methods formicroarray production are known to those of skill in the art and aredescribed in U.S. patents such as: U.S. Pat. Nos. 5,445,934; 5,532,128;5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186;5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531;5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711;5,658,734; or 5,700,637; each of which is herein incorporated byreference in its entirety. Other methods of profiling miRNAs aredescribed in Taylor et al., Gynecol Oncol. 2008 July; 110(1):13-21,Gilad et al, PLoS ONE. 2008 Sep. 5; 3(9):e3148, Lee et al., Annu RevPathol. 2008 September 25 and Mitchell et al, Proc Natl Acad Sci USA.2008 Jul. 29; 105(30):10513-8, Shen R et al, BMC Genomics. 2004 Dec. 14;5(1):94, Mina L et al, Breast Cancer Res Treat. 2007 June;103(2):197-208, Zhang L et al, Proc Natl Acad Sci USA. 2008 May 13;105(19):7004-9, Ross J S et al, Oncologist. 2008 May; 13(5):477-93,Schetter A J et al, JAMA. 2008 Jan. 30; 299(4):425-36, Staudt L M, NEngl J Med 2003; 348:1777-85, Mulligan G et al, Blood. 2007 Apr. 15;109(8):3177-88. Epub 2006 Dec. 21, McLendon R et al, Nature. 2008 Oct.23; 455(7216):1061-8, and U.S. Pat. Nos. 5,538,848, 5,723,591,5,876,930, 6,030,787, 6,258,569, and 5,804,375, each of which is hereinincorporated by reference.

Microarray technology allows for the measurement of the steady-statemRNA or miRNA levels of thousands of transcripts or miRNAssimultaneously thereby presenting a powerful tool for identifyingeffects such as the onset, arrest, or modulation of uncontrolled cellproliferation. Two microarray technologies, such as cDNA arrays andoligonucleotide arrays can be used. The product of these analyses aretypically measurements of the intensity of the signal received from alabeled probe used to detect a cDNA sequence from the sample thathybridizes to a nucleic acid sequence at a known location on themicroarray. Typically, the intensity of the signal is proportional tothe quantity of cDNA, and thus mRNA or miRNA, expressed in the samplecells. A large number of such techniques are available and useful.Methods for determining gene expression can be found in U.S. Pat. No.6,271,002 to Linsley, et al.; U.S. Pat. No. 6,218,122 to Friend, et al.;U.S. Pat. No. 6,218,114 to Peck et al.; or U.S. Pat. No. 6,004,755 toWang, et al., each of which is herein incorporated by reference in itsentirety.

Analysis of the expression levels is conducted by comparing suchintensities. This can be performed by generating a ratio matrix of theexpression intensities of genes in a test sample versus those in acontrol sample. The control sample may be used as a reference, anddifferent references to account for age, ethnicity and sex may be used.Different references can be used for different conditions or diseases,as well as different stages of diseases or conditions, as well as fordetermining therapeutic efficacy.

For instance, the gene expression intensities of mRNA or miRNAs isolatedfrom exosomes derived from a diseased tissue can be compared with theexpression intensities generated from exosomes isolated from normaltissue of the same type (e.g., diseased breast tissue sample versus.normal breast tissue sample). A ratio of these expression intensitiesindicates the fold-change in gene expression between the test andcontrol samples. Alternatively, if exosomes are not normally present infrom normal tissues (e.g. breast) then absolute quantitation methods, asis known in the art, can be used to define the number of miRNA moleculespresent without the requirement of miRNA or mRNA isolated from exosomesderived from normal tissue.

Gene expression profiles can also be displayed in a number of ways. Themost common method is to arrange raw fluorescence intensities or ratiomatrix into a graphical dendogram where columns indicate test samplesand rows indicate genes. The data is arranged so genes that have similarexpression profiles are proximal to each other. The expression ratio foreach gene is visualized as a color. For example, a ratio less than one(indicating down-regulation) may appear in the blue portion of thespectrum while a ratio greater than one (indicating up-regulation) mayappear as a color in the red portion of the spectrum. Commerciallyavailable computer software programs are available to display such data.

mRNAs or miRNAs that are considered differentially expressed can beeither over expressed or under expressed in patients with a diseaserelative to disease free individuals. Over and under expression arerelative terms meaning that a detectable difference (beyond thecontribution of noise in the system used to measure it) is found in theamount of expression of the mRNAs or miRNAs relative to some baseline.In this case, the baseline is the measured mRNA/miRNA expression of anon-diseased individual. The mRNA/miRNA of interest in the diseasedcells can then be either over or under expressed relative to thebaseline level using the same measurement method. Diseased, in thiscontext, refers to an alteration of the state of a body that interruptsor disturbs, or has the potential to disturb, proper performance ofbodily functions as occurs with the uncontrolled proliferation of cells.Someone is diagnosed with a disease when some aspect of that person'sgenotype or phenotype is consistent with the presence of the disease.However, the act of conducting a diagnosis or prognosis includes thedetermination of disease/status issues such as determining thelikelihood of relapse or metastasis and therapy monitoring. In therapymonitoring, clinical judgments are made regarding the effect of a givencourse of therapy by comparing the expression of genes over time todetermine whether the mRNA/miRNA expression profiles have changed or arechanging to patterns more consistent with normal tissue.

Levels of over and under expression are distinguished based on foldchanges of the intensity measurements of hybridized microarray probes. A2× difference is preferred for making such distinctions or a p-valueless than 0.05. That is, before an mRNA/miRNA is said to bedifferentially expressed in diseased/relapsing versusnormal/non-relapsing cells, the diseased cell is found to yield at least2 times more, or 2 times less intensity than the normal cells. Thegreater the fold difference, the more preferred is use of the gene as adiagnostic or prognostic tool. mRNA/miRNAs selected for the expressionprofiles of the instant invention have expression levels that result inthe generation of a signal that is distinguishable from those of thenormal or non-modulated genes by an amount that exceeds background usingclinical laboratory instrumentation.

Statistical values can be used to confidently distinguish modulated fromnon-modulated mRNA/miRNA and noise. Statistical tests find themRNA/miRNA most significantly different between diverse groups ofsamples. The Student's t-test is an example of a robust statistical testthat can be used to find significant differences between two groups. Thelower the p-value, the more compelling the evidence that the gene isshowing a difference between the different groups. Nevertheless, sincemicroarrays measure more than one mRNA/miRNA at a time, tens ofthousands of statistical tests may be performed at one time. Because ofthis, one is unlikely to see small p-values just by chance andadjustments for this using a Sidak correction as well as arandomization/permutation experiment can be made. A p-value less than0.05 by the t-test is evidence that the gene is significantly different.More compelling evidence is a p-value less then 0.05 after the Sidakcorrection is factored in. For a large number of samples in each group,a p-value less than 0.05 after the randomization/permutation test is themost compelling evidence of a significant difference.

In one embodiment, a method of generating a posterior probability scoreto enable diagnostic, prognostic, therapy-related, or physiologicalstate specific bio-signature scores can be arrived at by obtaining mRNAor miRNA (biomarker) expression data from a statistically significantnumber of patient exosomes, such as cell-of-origin specific exosomes;applying linear discrimination analysis to the data to obtain selectedbiomarkers; and applying weighted expression levels to the selectedbiomarkers with discriminate function factor to obtain a predictionmodel that can be applied as a posterior probability score. Otheranalytical tools can also be used to answer the same question such as,logistic regression and neural network approaches.

For instance, the following can be used for linear discriminantanalysis:

where,

I(p _(s) i _(d))=The log base 2 intensity of the probe set enclosed inparenthesis. d(cp)=The discriminant function for the disease positiveclass d(C _(N))=The discriminant function for the disease negative class

P(_(CP))=The posterior p-value for the disease positive class

P(_(CN))=The posterior p-value for the disease negative class

Numerous other well-known methods of pattern recognition are available.The following references provide some examples: Weighted Voting: Golubet al. (1999); Support Vector Machines: Su et al. (2001); and Ramaswamyet al. (2001); K-nearest Neighbors: Ramaswamy (2001); and CorrelationCoefficients: van 't Veer et al. (2002), all of which are hereinincorporated by reference in their entireties.

Bio-signature portfolios, further described below, can be establishedsuch that the combination of biomarkers in the portfolio exhibitimproved sensitivity and specificity relative to individual biomarkersor randomly selected combinations of biomarkers. In one embodiment, thesensitivity of the bio-signature portfolio can be reflected in the folddifferences, for example, exhibited by a transcript's expression in thediseased state relative to the normal state. Specificity can bereflected in statistical measurements of the correlation of thesignaling of transcript expression with the condition of interest. Forexample, standard deviation can be a used as such a measurement. Inconsidering a group of biomarkers for inclusion in a bio-signatureportfolio, a small standard deviation in expression measurementscorrelates with greater specificity. Other measurements of variationsuch as correlation coefficients can also be used in this capacity.

Another parameter that can be used to select mRNA/miRNA that generate asignal that is greater than that of the non-modulated mRNA/miRNA ornoise is the use of a measurement of absolute signal difference. Thesignal generated by the modulated mRNA/miRNA expression is at least 20%different than those of the normal or non-modulated gene (on an absolutebasis). It is even more preferred that such mRNA/miRNA produceexpression patterns that are at least 30% different than those of normalor non-modulated mRNA/miRNA.

MiRNA can also be detected and measured by amplification from abiological sample and measured using methods described in U.S. Pat. No.7,250,496, U.S. Application Publication Nos. 20070292878, 20070042380 or20050222399 and references cited therein, each of which is hereinincorporated by reference in its entirety.

Peptide nucleic acids (PNAs) which are a new class of synthetic nucleicacid analogs in which the phosphate-sugar polynucleotide backbone isreplaced by a flexible pseudo-peptide polymer may be utilized inanalysis of bio-signatures of exosomes. PNAs are capable of hybridizingwith high affinity and specificity to complementary RNA and DNAsequences and are highly resistant to degradation by nucleases andproteinases. Peptide nucleic acids (PNAs) are an attractive new class ofprobes with applications in cytogenetics for the rapid in situidentification of human chromosomes and the detection of copy numbervariation (CNV). Multicolor peptide nucleic acid-fluorescence in situhybridization (PNA-FISH) protocols have been described for theidentification of several human CNV-related disorders and infectiousdiseases. PNAs can also be utilized as molecular diagnostic tools tonon-invasively measure oncogene mRNAs with tumor targetedradionuclide-PNA-peptide chimeras. Methods of using PNAs are describedfurther in Pellestor F et al., Curr Pharm Des. 2008; 14(24):2439-44,Tian X et al, Ann N Y Acad. Sci. 2005 November, 1059:106-44, Paulasova Pand Pellestor F, Annales de Génétique, 47 (2004) 349-358, Stender H.Expert Rev Mol. Diagn. 2003 September; 3(5):649-55. Review, Vigneault etal., Nature Methods, 5(9), 777±779 (2008), each reference is hereinincorporated by reference in its entirety. These methods can be used toscreen the genetic materials isolated from exosomes. When applying thesetechniques to cell-of-origin specific exosomes they can be used toidentify a given molecular signal that directly pertains to the cell oforigin.

In addition, mutational analysis may be carried out for mRNAs and DNAthat are identified from the exosomes. For mutational analysis oftargets or biomarkers that are of RNA origin, the RNA (mRNA, miRNA orother) can be reverse transcribed into cDNA and subsequently sequencedor assayed for known SNPs (by Taqman SNP assays, for example), or singlenucleotide mutations, as well as using sequencing to look for insertionsor deletions to determine mutations present in the cell-of-origin.Muliplexed ligation dependent probe amplification (MLPA) couldalternatively be used for the purpose of identifying CNV in small andspecific areas of interest. For example, once the total RNA has beenobtained from isolated colon cancer-specific exosomes, cDNA can besynthesized and primers specific for exons 2 and 3 of the KRAS gene canbe used to amplify these two exons containing codons 12, 13 and 61 ofthe KRAS gene. The same primers used for PCR amplification can be usedfor Big Dye Terminator sequence analysis on the ABI 3730 to identifymutations in exons 2 and 3 of KRAS. Mutations in these codons are knownto confer resistance to drugs such as Cetuximab and Panitumimab. Methodsof conducting mutational analysis are described in Maheswaran S et al.,Jul. 2, 2008 (10.1056/NEJMoa0800668) and Orita, M et al, PNAS 1989,(86): 2766-70, each of which is herein incorporated by reference in itsentirety. Other methods of conducting mutational analysis can includemiRNA sequencing. Applications for identifying and profiling miRNAs canbe done by cloning techniques and the use of capillary DNA sequencing or“next-generation” sequencing technologies. The new sequencingtechnologies currently available allow the identification oflow-abundance miRNAs or those exhibiting modest expression differencesbetween samples, which may not be detected by hybridization-basedmethods. Such new sequencing technologies include the massively parallelsignature sequencing (MPSS) methodology described in Nakano et al. 2006,Nucleic Acids Res. 2006; 34:D731D735. doi: 10.1093/nar/gkj077, theRoche/454 platform described in Margulies et al. 2005, Nature. 2005;437:376 380 or the Illumina sequencing platform described in Berezikovet al. Nat. Genet. 2006b; 38:1375-1377, each of which is incorporated byreference in its entirety.

Additional methods to determine bio-signatures include assayingbiomarkers by allele-specific PCR which include specific primers toamplify and discriminate between two alleles of a gene simultaneously,single-strand conformation polymorphism (SSCP) which involves theelectrophoretic separation of single-stranded nucleic acids based onsubtle differences in sequence and DNA and RNA aptamers. DNA and RNAaptamers are short oligonucleotide sequences that can be selected fromrandom pools based on their ability to bind a particular molecule withhigh affinity. Methods of using aptamers are described in Ulrich H etal, Comb Chem High Throughput Screen. 2006 September; 9(8):619-32,Ferreira C S et al, Anal Bioanal Chem. 2008 February; 390(4):1039-50,Ferreira C S et al, Tumour Biol. 2006; 27(6):289-301, each of which isherein incorporated by reference in its entirety.

Exosome biomarkers can also be detected using fluorescence in situhybridization (FISH). Methods of using FISH to detect and localizespecific DNA sequences, localize specific mRNAs within tissue samples oridentify chromosomal abnormalities are described in Shaffer D R et al,Clin Cancer Res. 2007 Apr. 1; 13(7):2023-9, Cappuzo F et al, Journal ofThoracic Oncology, Volume 2, Number 5, May 2007, Moroni M et al., LancetOncol. 2005 May; 6(5):279-86, each of which is herein incorporated byreference in its entirety.

Bio-Signature: Binding Agents

Bio-signatures of exosomes can comprise binding agents for exosomes. Thebinding agent can be DNA, RNA, aptamers, monoclonal antibodies,polyclonal antibodies, Fabs, Fab′, single chain antibodies, syntheticantibodies, aptamers (DNA/RNA), peptoids, zDNA, peptide nucleic acids(PNAs), locked nucleic acids (LNAs), lectins, synthetic or naturallyoccurring chemical compounds (including but not limited to drugs,labeling reagents).

Binding agents can used to isolate exosomes by binding to exosomalcomponents, as described above. The binding agents can be used to detectthe exosomes, such as for detecting cell-of-origin specific exosomes. Abinding agent or multiple binding agents can themselves form a bindingagent profile that provides a bio-signature for an exosome. One or morebinding agents can be selected from FIG. 2. For example, if an exosomepopulation is detected or isolated using two, three or four bindingagents in a differential detection or isolation of an exosome from aheterogeneous population of exosomes, the particular binding agentprofile for the exosome population provides a bio-signature for theparticular exosome population.

As an illustrative example, an exosome for analysis for lung cancer canbe detected with one or more binding agents including, but not limitedto, SCLC specific aptamer HCA 12, SCLC specific aptamer HCC03, SCLCspecific aptamer HCH07, SCLC specific aptamer HCH01, A-p50 aptamer(NF-KB), Cetuximab, Panitumumab, Bevacizumab, L19 Ab, F16 Ab, anti-CD45(anti-ICAM-1, aka UV3), or L2G7 Ab (anti-HGF), or any combinationthereof.

An exosome for analysis for colon cancer can be detected with one ormore binding agents including, but not limited to, angiopoietin 2specific aptamer, beta-catenin aptamer, TCF1 aptamer, anti-Derlin1 ab,anti-RAGE, mAbgb3.1, Galectin-3, Cetuximab, Panitumumab, Matuzumab,Bevacizumab, or Mac-2, or any combination thereof.

An exosome for analysis for adenoma versus colorectal cancer (CRC) canbe detected with one or more binding agents including, but not limitedto, Complement C3, histidine-rich glycoprotein, kininogen-1, orGalectin-3, or any combination thereof.

An exosome for analysis for adenoma with low grade hyperplasia versusadenoma with high grade hyperplasia can be detected with a binding agentsuch as, but not limited to, Galectin-3 or any combination of bindingagents specific for this comparison.

An exosome for analysis for CRC versus normal state can be detected withone or more binding agents including, but not limited to, anti-ODC mAb,anti-CEA mAb, or Mac-2, or any combination thereof.

An exosome for analysis for prostate cancer can be detected with one ormore binding agents including, but not limited to, PSA, PSMA, TMPRSS2,mAB 5D4, XPSM-A9, XPSM-A 10, Galectin-3, E-selectin, Galectin-1, or E4(IgG2a kappa), or any combination thereof.

An exosome for analysis for melanoma can be detected with one or morebinding agents including, but not limited to, Tremelimumab (anti-CTLA4),Ipilimumumab (anti-CTLA4), CTLA-4 aptamers, STAT-3 peptide aptamers,Galectin-1, Galectin-3, or PNA, or any combination thereof.

An exosome for analysis for pancreatic cancer can be detected with oneor more binding agents including, but not limited to, H38-15 (anti-HGF)aptamer, H38-21(anti-HGF) aptamer, Matuzumab, Cetuximanb, orBevacizumab, or any combination thereof.

An exosome for analysis for brain cancer can be detected with one ormore binding agents including, but not limited to, aptamer III.1(pigpen) and/or TTA1 (Tenascin-C) aptamer, or any combination thereof.

An exosome for analysis for psoriasis can be detected with one or morebinding agents including, but not limited to, E-selectin, ICAM-1, VLA-4,VCAM-1, alphaEbeta7, or any combination thereof.

An exosome for analysis for cardiovascular disease (CVD) can be detectedwith one or more binding agents including, but not limited to, RB007(factor 1×A aptamer), ARC1779 (anti VWF) aptamer, or LOX1, or anycombination thereof.

An exosome for analysis for hematological malignancies can be detectedwith one or more binding agents including, but not limited to, anti-CD20and/or anti-CD52, or any combination thereof.

An exosome for analysis for B-cell chronic lymphocytic leukemias can bedetected with one or more binding agents including, but not limited to,Rituximab, Alemtuzumab, Apt48 (BCL6), R0-60, or D-R15-8, or anycombination thereof.

An exosome for analysis for B-cell lymphoma can be detected with one ormore binding agents including, but not limited to, Ibritumomab,Tositumomab, Anti-CD20 Antibodies, Alemtuzumab, Galiximab, Anti-CD40Antibodies, Epratuzumab, Lumiliximab, Hu1D10, Galectin-3, or Apt48, orany combination thereof.

An exosome for analysis for Burkitt's lymphoma can be detected with oneor more binding agents including, but not limited to, TD05 aptamer, IgMmAB (38-13), or any combination thereof.

An exosome for analysis for cervical cancer can be detected with one ormore binding agents including, but not limited to, Galectin-9 and/orHPVE7 aptamer, or any combination thereof.

An exosome for analysis for endometrial cancer can be detected with oneor more binding agents including, but not limited to, Galectin-1 or anycombinations of binding agents specific for endometrial cancer.

An exosome for analysis for head and neck cancer can be detected withone or more binding agents including, but not limited to, (111)In-cMAbU36, anti-LOXL4, U36, BIWA-1, BIWA-2, BIWA-4, or BIWA-8, or anycombination thereof.

An exosome for analysis for IBD can be detected with one or more bindingagents including, but not limited to, ACCA (anti-glycan Ab),ALCA(anti-glycan Ab), or AMCA (anti-glycan Ab), or any combinationthereof.

An exosome for analysis for diabetes can be detected with one or morebinding agents including, but not limited to, RBP4 aptamer or anycombination of binding agents specific for diabetes.

An exosome for analysis for fibromyalgia can be detected with one ormore binding agents including, but not limited to, L-selectin or anycombination of binding agents specific for fibromyalgia.

An exosome for analysis for multiple sclerosis (MS) can be detected withone or more binding agents including, but not limited to, Natalizumab(Tysabri) or any combination of binding agents specific for MS.

In addition, An exosome for analysis for rheumatic disease can bedetected with one or more binding agents including, but not limited to,Rituximab (anti-CD20 Ab) and/or Keliximab (anti-CD4 Ab), or anycombination of binding agents specific for rheumatic disease.

An exosome for analysis for Alzheimer disease can be detected with oneor more binding agents including, but not limited to, TH14-BACE1aptapers, S10-BACE1 aptapers, anti-Abeta, Bapineuzumab (AAB-001)-Elan,LY2062430 (anti-amyloid beta Ab)-Eli Lilly, or BACE1-Anti sense, or anycombination thereof.

An exosome for analysis for Prion specific diseases can be detected withone or more binding agents including, but not limited to, rhuPrP(c)aptamer, DP7 aptamer, Thioaptamer 97, SAF-93 aptamer, 15B3 (anti-PrPScAb), monoclonal anti PrPSc antibody P1:1, 1.5D7, 1.6F4 Abs, mab 14D3,mab 4F2, mab 8G8, or mab 12F10, or any combination thereof.

An exosome for analysis for sepsis can be detected with one or morebinding agents including, but not limited to, HA-1A mAb, E-5 mAb,TNF-alpha MAb, Afelimomab, or E-selectin, or any combination thereof.

An exosome for analysis for schizophrenia can be detected with one ormore binding agents including, but not limited to, L-selectin and/orN-CAM, or any combination of binding agents specific for schizophrenia.

An exosome for analysis for depression can be detected with one or morebinding agents including, but not limited to, GPIb or any combination ofbinding agents specific for depression.

An exosome for analysis for GIST can be detected with one or morebinding agents including, but not limited to, ANTI-DOG1 Ab or anycombination of binding agents specific for GIST.

An exosome for analysis for esophageal cancer can be detected with oneor more binding agents including, but not limited to, CaSR binding agentor any combination of binding agents specific for esophageal cancer.

An exosome for analysis for gastric cancer can be detected with one ormore binding agents including, but not limited to, Calpain nCL-2 bindingagent and/or drebrin binding agent, or any combination of binding agentsspecific for gastric cancer.

An exosome for analysis for COPD can be detected with one or morebinding agents including, but not limited to, CXCR3 binding agent, CCR5binding agent, or CXCR6 binding agent, or any combination of bindingagents specific for COPD.

An exosome for analysis for asthma can be detected with one or morebinding agents including, but not limited to, VIP binding agent, PACAPbinding agent, CGRP binding agent, NT3 binding agent, YKL-40 bindingagent, S-nitrosothiols, SCCA2 binding agent, PAI binding agent,amphiregulin binding agent, or Periostin binding agent, or anycombination of binding agents specific for asthma.

An exosome for analysis for vulnerable plaque can be detected with oneor more binding agents including, but not limited to, Gd-DTPA-g-mimRGD(Alpha v Beta 3 integrin binding peptide), or MMP-9 binding agent, orany combination of binding agents specific for vulnerable plaque.

An exosome for analysis for ovarian cancer can be detected with one ormore binding agents including, but not limited to, (90) Y-muHMFG1binding agent and/or OC125 (anti-CA125 antibody), or any combination ofbinding agents specific for ovarian cancer.

The binding agent can be for a general exosome marker, or “housekeepingprotein” or antigen, such as CD9, CD63, or CD81. For example, thebinding agent can be an antibody for CD9, CD63, or CD81. The bindingagent can also be for other exosomal proteins, such as for prostatespecific exosomes, or cancer specific exosomes, such as PCSA, PSMA,EpCam, B7H3, or STEAP. For example, the binding agent can be an antibodyfor PCSA, PSMA, EpCam, B7H3, or STEAP.

Furthermore, additional cellular binding partners or binding agents maybe identified by any conventional methods known in the art, or asdescribed herein, and may additionally be used as a diagnostic,prognostic or therapy-related marker.

Bio-Signatures: Prostate Cancer, Colon Cancer and Ovarian Cancer

Prostate Cancer

An exosome bio-signature can be used to characterize prostate cancer. Asdescribed above, a bio-signature for prostate cancer can comprise abinding agent associated with prostate cancer (for example, as shown inFIG. 2), and one or more additional biomarkers, such as shown in FIG.19. For example, a bio-signature for prostate cancer can comprise abinding agent to PSA, PSMA, TMPRSS2, mAB 5D4, XPSM-A9, XPSM-A10,Galectin-3, E-selectin, Galectin-1, E4 (IgG2a kappa), or any combinationthereof, with one or more additional biomarkers, such as one or moremiRNA, one or more DNA, one or more additional peptide, protein, orantigen associated with prostate cancer, such as, but not limited to,those shown in FIG. 19.

A bio-signature for prostate cancer can comprise an antigen associatedwith prostate cancer (for example, as shown in FIG. 1), and one or moreadditional biomarkers, such as shown in FIG. 19. A bio-signature forprostate cancer can comprise one or more antigens associated withprostate cancer, such as, but not limited to, KIA1, intact fibronectin,PSA, TMPRSS2, FASLG, TNFSF10, PSMA, NGEP, IL-7R1, CSCR4, CysLT1R, TRPM8,Kv1.3, TRPV6, TRPM8, PSGR, MISIIR, or any combination thereof. Thebio-signature for prostate cancer can comprise one or more of theaforementioned antigens and one or more additional biomarkers, such as,but not limited to miRNA, mRNA, DNA, or any combination thereof.

A bio-signature for prostate cancer can also comprise one or moreantigens associated with prostate cancer, such as, but not limited to,KIA1, intact fibronectin, PSA, TMPRSS2, FASLG, TNFSF10, PSMA, NGEP,IL-7R1, CSCR4, CysLT1R, TRPM8, Kv1.3, TRPV6, TRPM8, PSGR, MISIIR, or anycombination thereof, and one or more miRNA biomarkers, such as, but notlimited to, miR-202, miR-210, miR-296, miR-320, miR-370, miR-373,miR-498, miR-503, miR-184, miR-198, miR-302c, miR-345, miR-491, miR-513,miR-32, miR-182, miR-31, miR-26a-1/2, miR-200c, miR-375, miR-196a-1/2,miR-370, miR-425, miR-425, miR-194-1/2, miR-181a-1/2, miR-34b, let-71,miR-188, miR-25, miR-106b, miR-449, miR-99b, miR-93, miR-92-1/2,miR-125a, miR-141, let-7a, let-7b, let-7c, let-7d, let-7g, miR-16,miR-23a, miR-23b, miR-26a, miR-92, miR-99a, miR-103, miR-125a, miR-125b,miR-143, miR-145, miR-195, miR-199, miR-221, miR-222, miR-497, let-7f,miR-19b, miR-22, miR-26b, miR-27a, miR-27b, miR-29a, miR-29b,miR-30_(—)5p, miR-30c, miR-100, miR-141, miR-148a, miR-205, miR-520h,miR-494, miR-490, miR-133a-1, miR-1-2, miR-218-2, miR-220, miR-128a,miR-221, miR-499, miR-329, miR-340, miR-345, miR-410, miR-126, miR-205,miR-7-1/2, miR-145, miR-34a, miR-487, or let-7b, or any combinationthereof.

Furthermore, the miRNA for a prostate cancer bio-signature can be amiRNA that interacts with PFKFB3, RHAMM (HMMR), cDNA FLJ42103, ASPM,CENPF, NCAPG, Androgen Receptor, EGFR, HSP90, SPARC, DNMT3B, GART, MGMT,SSTR3, TOP2B, or any combination thereof, such as those described hereinand depicted in FIG. 60. The miRNA can also be miR-9, miR-629, miR-141,miR-671-3p, miR-491, miR-182, miR-125a-3p, miR-324-5p, miR-148B,miR-222, or any combination thereof.

The bio-signature for prostate cancer can comprise one or more antigensassociated with prostate cancer, such as, but not limited to, KIA1,intact fibronectin, PSA, TMPRSS2, FASLG, TNFSF10, PSMA, NGEP, IL-7R1,CSCR4, CysLT1R, TRPM8, Kv1.3, TRPV6, TRPM8, PSGR, MISIIR, or anycombination thereof, and one or more additional biomarkers such as, butnot limited to, the aforementioned miRNAs, mRNAs (such as, but notlimited to, AR or PCA3), snoRNA (such as, but not limited to, U50) orany combination thereof.

The bio-signature can also comprise one or more gene fusions, such asACSL3-ETV 1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV 1, TMPRSS2-ERG,TMPRSS2-ETV 1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5or KLK2-ETV4.

An exosome can be isolated and assayed for one or more miRNA and one ormore antigens associated with prostate cancer to provide a diagnostic,prognostic or theranostic profile, such as the stage of the cancer, theefficacy of the cancer, or other characteristics of the cancer.Alternatively, the exosome can be directly assayed from a sample, suchthat the exosomes are not purified or concentrated prior to assaying forone or more miRNA or antigens associated with prostate cancer.

As depicted in FIG. 68, a prostate cancer bio-signature can compriseassaying EpCam, CD63, CD81, CD9, or any combination thereof, of anexosome. The prostate cancer bio-signature can comprise detection ofEpCam, CD9, CD63, CD81, PCSA or any combination thereof. For example,the prostate cancer bio-signature can comprise EpCam, CD9, CD63 and CD81or PCSA, CD9, CD63 and CD81 (see for example, FIG. 70A). The prostatecancer bio-signature can also comprise PCSA, PSMA, B7H3, or anycombination thereof (see for example, FIG. 70B).

Furthermore, assessing a plurality of biomarkers can provide increasedsensitivity, specificity, or signal intensity, as compared to assessingless than a plurality of biomarkers. For example, assessing PSMA andB7H3 can provide increased sensitivity in detection as compared toassessing PSMA or B7H3 alone. Assessing CD9 and CD63 can provideincreased sensitivity in detection as compared to assessing CD or CD63alone.

Prostate cancer can also be characterized based on meeting at least 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 criteria. For example, a number ofdifferent criteria can be used: 1) if the amount of exosomes in a samplefrom a subject is higher than a reference value; 2) if the amount ofprostate cell derived exosomes is higher than a reference value; and 3)if the amount of exosomes with one or more cancer specific biomarkers ishigher than a reference value, the subject is diagnosed with prostatecancer. The method can further include a quality control measure, suchthat the subject is diagnosed with prostate cancer if the determinationof the other criteria

The prostate cancer can be characterizing using one or more processesdisclosed herein with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,or 70% sensitivity. The prostate cancer can be characterized with atleast 80, 81, 82, 83, 84, 85, 86, or 87% sensitivity. For example, theprostate cancer can be characterized with at least 87.1, 87.2, 87.3,87.4, 87.5, 87.6, 87.7, 87.8, 87.9, 88.0, or 89% sensitivity, such aswith at least 90% sensitivity, such as at least 91, 92, 93, 94, 95, 96,97, 98, 99 or 100% sensitivity.

The prostate cancer of a subject can also be characterized with at least70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, or 97% specificity, such as with atleast 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0,98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% specificity.

The prostate cancer can also be characterized with at least 70%sensitivity and at least 80, 90, 95, 99, or 100% specificity; at least80% sensitivity and at least 80, 85, 90, 95, 99, or 100% specificity; atleast 85% sensitivity and at least 80, 85, 90, 95, 99, or 100%specificity; at least 86% sensitivity and at least 80, 85, 90, 95, 99,or 100% specificity; at least 87% sensitivity and at least 80, 85, 90,95, 99, or 100% specificity; at least 88% sensitivity and at least 80,85, 90, 95, 99, or 100% specificity; at least 89% sensitivity and atleast 80, 85, 90, 95, 99, or 100% specificity; at least 90% sensitivityand at least 80, 85, 90, 95, 99, or 100% specificity; at least 95%sensitivity and at least 80, 85, 90, 95, 99, or 100% specificity; atleast 99% sensitivity and at least 80, 85, 90, 95, 99, or 100%specificity; or at least 100% sensitivity and at least 80, 85, 90, 95,99, or 100% specificity.

Furthermore, the confidence level for determining the specificity,sensitivity, or both, may be with at least 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% confidence.

Colon Cancer

A colon cancer bio-signature can comprise any one or more antigens forcolon cancer as listed in FIG. 1, any one or more binding agentsassociated with isolating an exosome for characterizing colon cancer(for example, as shown in FIG. 2), any one or more additionalbiomarkers, such as shown in FIG. 6.

The bio-signature can comprise one or more miRNA selected from the groupconsisting of miR-24-1, miR-29b-2, miR-20a, miR-10a, miR-32, miR-203,miR-106a, miR-17-5p, miR-30c, miR-223, miR-126, miR-128b, miR-21,miR-24-2, miR-99b, miR-155, miR-213, miR-150, miR-107, miR-191, miR-221,miR-20a, miR-510, miR-92, miR-513, miR-19a, miR-21, miR-20, miR-183,miR-96, miR-135b, miR-31, miR-21, miR-92, miR-222, miR-181b, miR-210,miR-20a, miR-106a, miR-93, miR-335, miR-338, miR-133b, miR-346,miR-106b, miR-153a, miR-219, miR-34a, miR-99b, miR-185, miR-223,miR-211, miR-135a, miR-127, miR-203, miR-212, miR-95, or miR-17-5p, orany combination thereof. The bio-signature can also comprise one or moreunderexpressed miRs such as miR-143, miR-145, miR-143, miR-126, miR-34b,miR-34c, let-7, miR-9-3, miR-34a, miR-145, miR-455, miR-484, miR-101,miR-145, miR-133b, miR-129, miR-124a, miR-30-3p, miR-328, miR-106a,miR-17-5p, miR-342, miR-192, miR-1, miR-34b, miR-215, miR-192, miR-301,miR-324-5p, miR-30a-3p, miR-34c, miR-331, and miR-148b.

The bio-signature can comprise assessing one or more genes, such asEFNB1, ERCC1, HER2, VEGF, and EGFR. A biomarker mutation for coloncancer that can be assessed in an exosome can also include one or moremutations of EGFR, KRAS, VEGFA, B-Raf, APC, or p53. The bio-signaturecan also comprise one or more proteins, ligands, or peptides that can beassessed of an exosome such as AFRs, Rabs, ADAM10, CD44, NG2, ephrin-B1,MIF, b-catenin, Junction, plakoglobin, glalectin-4, RACK1, tetrspanin-8,FasL, TRAIL, A33, CEA, EGFR, dipeptidase 1, hsc-70, tetraspanins, ESCRT,TS, PTEN, or TOPO1

An exosome can be isolated and assayed for to provide a diagnostic,prognostic or theranostic profile, such as the stage of the cancer, theefficacy of the cancer, or other characteristics of the cancer.Alternatively, the exosome can be directly assayed from a sample, suchthat the exosomes are not purified or concentrated prior to assaying fora bio-signature associated with colon cancer.

As depicted in FIG. 69, a colon cancer signature can comprise detectionof EpCam, CD63, CD81, CD9, CD66, or any combination thereof, of anexosome. Furthermore, a colon cancer-bio-signature for various stages ofcancer can comprise CD63, CD9, EpCam, or any combination thereof (seefor example, FIGS. 71 and 72). For example, the bio-signature cancomprise CD9 and EpCam.

The colon cancer can be characterizing using one or more processesdisclosed herein with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,or 70% sensitivity. The colon cancer can be characterized with at least80, 81, 82, 83, 84, 85, 86, or 87% sensitivity. For example, the coloncancer can be characterized with at least 87.1, 87.2, 87.3, 87.4, 87.5,87.6, 87.7, 87.8, 87.9, 88.0, or 89% sensitivity, such as with at least90% sensitivity, such as at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or100% sensitivity.

The colon cancer of a subject can also be characterized with at least70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, or 97% specificity, such as with atleast 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0,98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% specificity.

The colon cancer can also be characterized with at least 70% sensitivityand at least 80, 90, 95, 99, or 100%) specificity; at least 80%sensitivity and at least 80, 85, 90, 95, 99, or 100% specificity; atleast 85% sensitivity and at least 80, 85, 90, 95, 99, or 100%specificity; at least 86% sensitivity and at least 80, 85, 90, 95, 99,or 100% specificity; at least 87% sensitivity and at least 80, 85, 90,95, 99, or 100% specificity; at least 88% sensitivity and at least 80,85, 90, 95, 99, or 100% specificity; at least 89% sensitivity and atleast 80, 85, 90, 95, 99, or 100% specificity; at least 90% sensitivityand at least 80, 85, 90, 95, 99, or 100% specificity; at least 95%sensitivity and at least 80, 85, 90, 95, 99, or 100% specificity; atleast 99% sensitivity and at least 80, 85, 90, 95, 99, or 100%specificity; or at least 100% sensitivity and at least 80, 85, 90, 95,99, or 100% specificity.

Furthermore, the confidence level for determining the specificity,sensitivity, or both, may be with at least 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% confidence.

Ovarian Cancer

A bio-signature for characterizing ovarian cancer can comprise anantigen associated with ovarian cancer (for example, as shown in FIG.1), and one or more additional biomarkers, such as shown in FIG. 4.

In one embodiment, a bio-signature for ovarian cancer can comprise oneor more antigens associated with ovarian cancer, such as, but notlimited to, CD24, CA125, VEGF1, VEGFR2, HER2, MISIIR, or any combinationthereof. The bio-signature for ovarian cancer can comprise one or moreof the aforementioned antigens and one or more additional biomarker,such as, but not limited to miRNA, mRNA, DNA, or any combinationthereof. The bio-signature for ovarian cancer can comprise one or moreantigens associated with ovarian cancer, such as, but not limited to,CD24, CA125, VEGF I, VEGFR2, HER2, MISIIR; or any combination thereof,with one or more miRNA biomarkers, such as, but not limited to,miR-200a, miR-141, miR-200c, miR-200b, miR-21, miR-141, miR-200a,miR-200b, miR-200c, miR-203, miR-205, miR-214, miR-215, miR-199a,miR-140, miR-145, miR-125b-1, or any combination thereof.

A bio-signature for ovarian cancer can comprise one or more antigensassociated with ovarian cancer, such as, but not limited to, CD24,CA125, VEGF1, VEGFR2, HER2, MISIIR, or any combination thereof, with oneor more miRNA biomarkers (such as the aforementioned miRNA), mRNAs (suchas, but not limited to, ERCC1, ER, TOPO1, TOP2A, AR, PTEN, HER2/neu,EGFR), mutations (including, but not limited to, those relating to KRASand/or B-Raf) or any combination thereof.

An exosome can be isolated and assayed for one or more miRNA and one ormore antigens associated with ovarian cancer to provide a diagnostic,prognostic or theranostic profile. Alternatively, the exosome can bedirectly assayed from a sample, such that the exosomes are not purifiedor concentrated prior to assaying for one or more miRNA or antigensassociated with ovarian cancer.

Bio-Signatures: Assessing Organ Transplant Rejection and AutoimmuneConditions

An exosome can also be used for determining phenotypes such as organdistress and/or organ transplant rejection. As used herein organtransplant includes partial organ or tissue transplant. The presence,absence or levels of one or more biomarkers present in exosomes isassessed to monitor organ rejection or success. The level, or amount, ofexosomes in the sample can also be used to assess organ rejection orsuccess. The assessment can be determined with at least 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%specificity, sensitivity, or both. For example, the assessment can bedetermined with at least 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1,98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 998.2, 99.3,99.4, 99.5, 99.6, 99.7, 99.8, 99.9% sensitivity, specificity, or both

The exosome can be purified or concentrated prior to analysis.Alternatively, the level, or amount, of exosomes can be directly assayedfrom a sample, without prior purification or concentration. The exosomequantitated can be a cell-of-origin specific exosome. For example, acell or tissue-specific exosome can be isolated using one or morebinding agents specific for a particular organ. The cell-of-originspecific exosome can be assessed for one or more molecular features,such as one or more biomarkers associated with organ distress or organtransplant rejection. The presence, absence or levels of one or morebiomarkers present in an isolated cell-of-origin specific exosome can beassessed to monitor organ rejection or success.

One or more exosomes can be analyzed for the assessment, detection ordiagnosis of the rejection of a tissue or organ transplant by a subject.The tissue or organ transplant rejection can be hyperacute, acute, orchronic rejection. The exosome can also be analyzed for the assessment,detection or diagnosis of graft versus host disease in a subject. Thesubject can be the recipient of an autogenic, allogenic or xenogenictissue or organ transplant.

The exosome can also be analyzed to detect the rejection of a tissue ororgan transplant. The exosome may be produced by the tissue or organtransplant. Such tissues or organs include, but are not limited to, aheart, lung, pancreas, kidney, eye, cornea, muscle, bone marrow, skin,cartilage, bone, appendages, hair, face, tendon, stomach, intestine,vein, artery, differentiated cells, partially differentiated cells orstem cells.

The exosome can comprise at least one biomarker which is used to assess,diagnose or determine the probability or occurrence of rejection of atissue or organ transplant by a subject. A biomarker can also be used toassess, diagnose or detect graft versus host disease in a subject. Thebiomarker can be a protein, a polysaccharide, a fatty acid or a nucleicacid (such as DNA or RNA). The biomarker can be associated with therejection of a specific tissue or organ or systemic organ failure. Morethan one biomarker can be analyzed, for example, one or more proteinsmarker can be analyzed in combination with one or more nucleic acidmarkers. The biomarker may be an intracellular or extracellular marker.

The exosome can also be analyzed for at least one marker for theassessment, detection or diagnosis of cell apoptosis or necrosisassociated with, or the causation of, rejection of a tissue or organtransplant by a subject.

The presence of a biomarker can be indicative of the rejection of atissue or an organ by a subject, wherein the biomarker includes, but isnot limited to, CD40, CD40 ligand, N-acetylmuramoyl-L-alanine amidaseprecursor, adiponectin, AMBP protein precursor, C4b-binding proteina-chain precursor, ceruloplasmin precursor, complement C3 precursor,complement component C9 precursor, complement factor D precursor, alpha1-B-glycoprotein, beta2-glycoprotein I precursor, heparin cofactor IIprecursor, Immunoglobulin mu chain C region protein, Leucine-richalpha2-glycoprotein precursor, pigment epithelium-derived factorprecursor, plasma retinol-binding protein precursor, translationinitiation factor 3 subunit 10, ribosomal protein L7, beta-transducin,1-TRAF, or lysyl-tRNA synthetase.

Rejection of a kidney by a subject can also be detected by analyzingexosomes for the presence of beta-transducin. Rejection of transplantedtissue can also be detected by isolating a cell-of-origin specificexosome from CD40-expressing cells and detecting for the increase ofBcl-2 or TNFalpha.

Rejection of a liver transplant by a subject can be detected byanalyzing the exosomes for the presence of an F1 antigen marker. The F1antigen is, without being bound to theory, specific to liver to and canbe used to detect an increase in liver cell-of-origin specific exosomes.This increase can be used as an early indication of organdistress/rejection.

Bronchiolitis obliterans due to bone marrow and/or lung transplantationor other causes, or graft atherosclerosis/graft phlebosclerosis can alsobe diagnosed by the analysis of an exosome.

An exosome can also be analyzed for the detection, diagnosis orassessment of an autoimmune or other immunological reaction-relatedphenotypes in a subject. Examples of such a disorder include, but arenot limited to, systemic lupus erythematosus (SLE), discoid lupus, lupusnephritis, sarcoidosis, inflammatory arthritis, including juvenilearthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome,ankylosing spondylitis, and gouty arthritis, multiple sclerosis, hyperIgE syndrome, polyarteritis nodosa, primary biliary cirrhosis,inflammatory bowel disease, Crohn's disease, celiac's disease(gluten-sensitive enteropathy), autoimmune hepatitis, pernicious anemia,autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis,autoimmune thrombocytopenic purpura, autoimmune thyroiditis, Grave'sdisease, Hasimoto's thyroiditis, immune complex disease, chronic fatigueimmune dysfunction syndrome (CFIDS), polymyositis and dermatomyositis,cryoglobulinemia, thrombolysis, cardiomyopathy, pemphigus vulgaris,pulmonary interstitial fibrosis, asthma, Churg-Strauss syndrome(allergic granulomatosis), atopic dermatitis, allergic and irritantcontact dermatitis, urtecaria, IgE-mediated allergy, atherosclerosis,vasculitis, idiopathic inflammatory myopathies, hemolytic disease,Alzheimer's disease, chronic inflammatory demyelinating polyneuropathyand AIDs.

One or more biomarkers from the exosome can be used to assess, diagnoseor determine the probability of the occurrence of an autoimmune or otherimmunological reaction-related disorder in a subject. The biomarker canbe a protein, a polysaccharide, a fatty acid or a nucleic acid (such asDNA or RNA). The biomarker can be associated with a specific autoimmunedisorder, a systemic autoimmune disorder, or other immunologicalreaction-related disorder. More than one biomarker can be analyzed. Forexample one or more protein markers can be analyzed in combination withone or more nucleic acid markers. The biomarker can be an intracellularor extracellular marker. The biomarker can also be used to detect,diagnose or assess inflammation.

Analysis of an exosome from subjects can be used identify subjects withinflammation associated with asthma, sarcoidosis, emphysema, cysticfibrosis, idiopathic pulmonary fibrosis, chronic bronchitis, allergicrhinitis and allergic diseases of the lung such as hypersensitivitypneumonitis, eosinophilic pneumonia, as well as pulmonary fibrosisresulting from collagen, vascular, and autoimmune diseases such asrheumatoid arthritis.

Exosome Compositions

Also provided herein is an isolated exosome with a particularbio-signature. The isolated exosome can comprise one or more biomarkersor bio-signatures specific for specific cell type, or for characterizinga phenotype, such as described above. For example, the isolated exosomecan comprise one or more biomarkers, such as CD63, EpCam, CD81, CD9,PCSA, PSMA, B7H3, TNFR, MFG-E8, Rab, STEAP, 5T4, or CD59. The isolatedexosome can have the one or more biomarkers on its surface of within theexosome. The isolated exosome can also comprise one or more miRNAs, suchas miR-9, miR-629, miR-141, miR-671-3p, miR-491, miR-182, miR-125a-3p,miR-324-5p, miR-148B, or miR-222. An isolated exosome can comprise abiomarker such as CD66, and further comprise one or more biomarkersselected from the group consisting of: EpCam, CD63, or CD9. An isolatedexosome can also comprise a fusion gene or protein, such as TMRSSG2:ERG.

An isolated exosome can also comprise one or more biomarkers, whereinthe expression level of the one or more biomarkers is higher, lower, orthe same for an isolated exosome as compared to an isolated exosomederived from a normal cell (ie. a cell derived from a subject without aphenotype of interest). For example, an isolated exosome can compriseone or more biomarkers selected from the group consisting of: B7H3,PSCA, MFG-E8, Rab, STEAP, PSMA, PCSA, 5T4, miR-9, miR-629, miR-141,miR-671-3p, miR-491, miR-182, miR-125a-3p, miR-324-5p, miR-148b, andmiR-222, wherein the expression level of the one or more biomarkers ishigher for an isolated exosome as compared to an isolated exosomederived from a normal cell. The isolated exosome can comprise at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, or 19 of thebiomarkers selected from the group. The isolated exosome can furthercomprising one or more biomarkers selected from the group consisting of:EpCam, CD63, CD59, CD81, or CD9.

An isolated exosome can comprise the biomarkers PCSA, EpCam, CD63, andCD8; the biomarkers PCSA, EpCam, B7H3 and PSMA. An isolated exosome cancomprise the biomarkers miR-9, miR-629, miR-141, miR-671-3p, miR-491,miR-182, miR-125a-3p, miR-324-5p, miR-148b, and miR-222.

A composition comprising an isolated exosome is also provided herein.The composition can comprise one or more isolated exosomes. For example,the composition can comprise a plurality of exosomes, or one or morepopulations of exosomes.

The composition can be substantially enriched for exosomes. For example,the composition can be substantially absent of cellular debris, cells,or non-exosomal proteins, peptides, or nucleic acids (such as biologicalmolecules not contained within the exosomes). The cellular debris,cells, or non-exosomal proteins, peptides, or nucleic acids, can bepresent in a biological sample along with exosomes. A composition can besubstantially absent of cellular debris, cells, or non-exosomalproteins, peptides, or nucleic acids (such as biological molecules notcontained within the exosomes), can be obtained by any method disclosedherein, such as through the use of one or more binding agents or captureagents for one or more exosomes. The exosomes can comprise at least 30,40, 50, 60, 70, 80, 90, 95 or 99% of the total composition, by weight orby mass. The exosomes of the composition can be a heterogeneous orhomogeneous population of exosomes. For example, a homogeneouspopulation of exosomes comprises exosomes that are homogeneous as to oneor more properties or characteristics. For example, the one or morecharacteristics can be selected from a group consisting of: one or moreof the same biomarkers, a substantially similar or identicalbio-signature, derived from the same cell type, exosomes of a particularsize, and a combination thereof.

Thus, in some embodiments, the composition comprises a substantiallyenriched population of exosomes. The composition can be enriched for apopulation of exosomes that are at least 30, 40, 50, 60, 70, 80, 90, 95or 99% homogeneous as to one or more properties or characteristics. Forexample, the one or more characteristics can be selected from a groupconsisting of: one or more of the same biomarkers, a substantiallysimilar or identical bio-signature, derived from the same cell type,exosomes of a particular size, and a combination thereof. For example,the population of exosomes can be homogeneous by all having a particularbio-signature, having the same biomarker, having the same biomarkercombination, or derived from the same cell type. In some embodiments,the composition comprises a substantially homogeneous population ofexosomes, such as a population with a specific bio-signature, derivedfrom a specific cell, or both.

The population of exosome can comprise one or more of the samebiomarkers. The biomarker can be any component present in an exosome oron the exosome, such as any nucleic acid (e.g. RNA or DNA), protein,peptide, polypeptide, antigen, lipid, carbohydrate, or proteoglycan. Forexample, each exosome in a population can comprise the same or identicalone or more biomarkers. In some embodiments, each exosome in thepopulation comprises the same 1, 2, 3, 4, 5, 6, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75 or 100 biomarkers.The one or more biomarkers can be selected from FIGS. 1, 3-60.

The exosome population comprising the same or identical biomarker canrefer to each exosome in the population having the same presence orabsence, expression level, mutational state, or modification of thebiomarker. For example, an enriched population of exosome can compriseexosomes, wherein each exosome has the same biomarker present, the samebiomarker absent, the same expression level of a biomarker, the samemodification of a biomarker, or the same mutation of a biomarker. Thesame expression level of a biomarker can refer to a quantitative orqualitive measurement, such as the exosomes in the populationunderexpress, overexpress, or have the same expression level of abiomarker as compared to a reference level. Alternatively, the sameexpression level of a biomarker can be a numerical value representingthe expression of a biomarker that is similar for each exosome in apopulation. For example the copy number of a miRNA, the amount ofprotein, or the level of mRNA of each exosome can be quantitativelysimilar for each exosome in a population, such that the numerical amountof each exosome is ±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20% from theamount in each other exosome in the population, as such variations areappropriate.

In some embodiments, the composition comprises a substantially enrichedpopulation of exosomes, wherein the exosomes in the enriched populationhas a substantially similar or identical bio-signature. Thebio-signature can comprise one or more exosomal characteristic such asthe level or amount of exosomes, temporal evaluation of the variation inexosomal half-life, circulating exosomal half-life or exosomal metabolichalf-life, or the activity of an exosome. The bio-signature can alsocomprise the presence or absence, expression level, mutational state, ormodification of a biomarker, such as those described herein.

The bio-signature of each exosome in the population can be at least 30,40, 50, 60, 70, 80, 90, 95, or 99% identical. In some embodiments, thebio-signature of each exosome is 100% identical. The bio-signature ofeach exosome in the enriched population can have the same 1, 2, 3, 4, 5,6, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,50, 75 or 100 exosomal characteristics. For example, a bio-signature ofan exosome in an enriched population can be the presence of a firstbiomarker, the presence of a second biomarker, and the underexpressionof a third biomarker. Another exosome in the same population can be 100%identical, having the same first and second biomarkers present andunderexpression of the third biomarker. Alternatively, an exosome in thesame population can have the same first and second biomarkers, but nothave underexpression of the third biomarker.

In some embodiments, the composition comprises a substantially enrichedpopulation of exosomes, wherein the exosomes are derived from the samecell type. For example, the exosomes can all be derived from cells of aspecific tissue, cells from a specific tumor of interest or a diseasedtissue of interest, circulating tumor cells, or cells of maternal orfetal origin. The exosomes can all be derived from tumor cells. Theexosomes can all be derived from lung, pancreas, stomach, intestine,bladder, kidney, ovary, testis, skin, colorectal, breast, prostate,brain, esophagus, liver, placenta, or fetal cells.

The composition comprising a substantially enriched population ofexosomes can also comprise exosomes are of a particular size. Forexample, the exosomes can all a diameter of greater than about 10, 20,or 30 nm. They can all have a diameter of about 30-1000 nm, about 30-800nm, about 30-200 nm, or about 30-100 nm. In some embodiments, theexosomes can all have a diameter of less than about 10,000 nm, 1000 nm,800 nm, 500 nm, 200 nm, 100 nm or 50 nm.

The population of exosomes homogeneous for one or more characteristicscan comprises at least about 30, 40, 50, 60, 70, 80, 90, 95, or 99% ofthe total exosome population of the composition. In some embodiments, acomposition comprising a substantially enriched population of exosomescomprises at least 2, 3, 4, 5, 10, 20, 25, 50, 100, 250, 500, or 1000times the concentration of an exosome as compared to a concentration ofthe exosome in a biological sample from which the composition wasderived. In yet other embodiments, the composition can further comprisea second enriched population of exosomes, wherein the population ofexosomes is at least 30% homogeneous as to one or more characteristics,as described herein.

Multiplex analysis can be used to obtain a composition substantiallyenriched for more than one population of exosomes, such as at least 2,3, 4, 5, 6, 7, 8, 9, 10 exosome populations. Each substantially enrichedexosome population can comprise at least 5, 10, 15, 20, 25, 30, 35, 40,45, 46, 47, 48, or 49% of the composition, by weight or by mass. In someembodiments, the substantially enriched exosome populations comprises atleast about 30, 40, 50, 60, 70, 80, 90, 95, or 99% of the composition,by weight or by mass.

A substantially enriched population of exosomes can be obtained by usingone or more methods, processes, or systems as disclosed herein. Forexample, isolation of a population of exosomes from a sample can beperformed by using one or more binding agents for one or more biomarkersof an exosome, such as using two or more binding agents that target twoor more biomarkers of an exosome. One or more capture agents can be usedto obtain a substantially enriched population of exosomes. One or moredetection agents can be used to identify a substantially enrichedpopulation of exosomes.

In one embodiment, a population of exosomes with a particularbio-signature is obtained by using one or more binding agents for thebiomarkers of the bio-signature. The exosomes can be isolated resultingin a composition comprising a substantially enriched population ofexosomes with the particular bio-signature. In another embodiment, apopulation of exosomes with a particular bio-signature of interest canbe obtained by using one or more binding agents for biomarkers that arenot a component of the bio-signature of interest. Thus, the bindingagents can be used to remove the exosomes that do not have thebio-signature of interest and the resulting composition is substantiallyenriched for the population of exosomes with the particularbio-signature of interest. The resulting composition can besubstantially absent of the exosomes comprising a biomarker for thebinding agent.

Detection System and Kits

Also provided is a detection system configured to determine one or morebio-signatures for an exosome. The detection system can be used todetect a heterogeneous population of exosomes or one or more homogeneouspopulation of exosomes. The detection system can be configured to detecta plurality of exosomes, wherein at least a subset of said plurality ofexosomes comprises a different bio-signature from another subset of saidplurality of exosomes. The detection system detect at least 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 differentsubsets of exosomes, wherein each subset of exosomes comprises adifferent bio-signature. For example, a detection system, such as usingone or more methods, processes, and compositions disclosed herein, canbe used to detect at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, or 100 different populations of exosomes.

The detection system can be configured to assess at least 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 2500,5000, 7500, 10,000, 100,000, 150,000, 200,000, 250,000, 300,000,350,000, 400,000, 450,000, 500,000, 750,000, or 1,000,000 differentbiomarkers for one or more exosomes. In some embodiments, the one ormore biomarkers are selected from FIGS. 1, 3-60, or as disclosed herein.The detection system can be configured to assess a specific populationof exosomes, such as exosomes from a specific cell-of-origin, or toassess a plurality of specific populations of exosomes, wherein eachpopulation of exosomes has a specific bio-signature.

The detection system can be a low density detection system or a highdensity detection system. For example, a low density detection systemcan detect up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different exosomepopulations, whereas a high density detection system can detect at leastabout 15, 20, 25, 50, or 100 different exosome populations In anotherembodiment, a low density detection system can detect up to about 100,200, 300, 400, or 500 different biomarkers, whereas a high densitydetection system can detect at least about 750, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9,000, 10,000, 15,000, 20,000, 25,000, 50,000,or 100,000 different biomarkers. In yet another embodiment, a lowdensity detection system can detect up to about 100, 200, 300, 400, or500 different bio-signatures or biomarker combinations, whereas a highdensity detection system can detect at least about 750, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9,000, 10,000, 15,000, 20,000,25,000, 50,000, or 100,000 bio-signatures or biomarker combinations.

The detection system can comprise a probe that selectively hybridizes toan exosome. The detection system can comprise a plurality of probes todetect an exosome. In some embodiments, a plurality of probes is used todetect the amount of exosomes in a heterogeneous population of exosomes.In yet other embodiments, a plurality of probes is used to detect ahomogeneous population of exosomes. A plurality of probes can be used toisolate or detect at least two different subsets of exosomes, whereineach subset of exosomes comprises a different bio-signature.

A detection system, such as using one or more methods, processes, andcompositions disclosed herein, can comprise a plurality of probesconfigured to detect, or isolate, such as using one or more methods,processes, and compositions disclosed herein at least 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 differentsubsets of exosomes, wherein each subset of exosomes comprises adifferent bio-signature.

For example, a detection system can comprise a plurality of probesconfigured to detect at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 40, 50, 60, 70, 80, 90, or 100 different populations of exosomes.The detection system can comprise a plurality of probes configured toselectively hybridize to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 2500, 5000, 7500, 10,000,100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000,500,000, 750,000, or 1,000,000 different biomarkers for one or moreexosomes. In some embodiments, the one or more biomarkers are selectedfrom FIGS. 1, 3-60, or as disclosed herein. The plurality of probes canbe configured to assess a specific population of exosomes, such asexosomes from a specific cell-of-origin, or to assess a plurality ofspecific populations of exosomes, wherein each population of exosomeshas a specific bio-signature.

The detection system can be a low density detection system or a highdensity detection system comprising probes to detect exosomes. Forexample, a low density detection system can comprise probes to detect upto 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different exosome populations,whereas a high density detection system can comprise probes to detect atleast about 15, 20, 25, 50, or 100 different exosome populations Inanother embodiment, a low density detection system can comprise probesto detect up to about 100, 200, 300, 400, or 500 different biomarkers,whereas a high density detection system can comprise probes to detect atleast about 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9,000,10,000, 15,000, 20,000, 25,000, 50,000, or 100,000 different biomarkers.In yet another embodiment, a low density detection system can compriseprobes to detect up to about 100, 200, 300, 400, or 500 differentbio-signatures or biomarker combinations, whereas a high densitydetection system can comprise probes to detect at least about 750, 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9,000, 10,000, 15,000, 20,000,25,000, 50,000, or 100,000 bio-signatures or biomarker combinations.

The probes can be specific for detecting a specific exosome population,for example an exosome with a particular bio-signature, and as describedabove. A plurality of probes for detecting prostate specific exosomes isalso provided. A plurality of probes can comprise probes for detectingone or more of the following biomarkers: CD9, PSCA, TNFR, CD63, MFG-E8,EpCAM, Rab, CD81, STEAP, PCSA, 5T4, EpCAM, PSMA, CD59, CD66, CD24 andB7H3. A plurality of probes for detecting Bcl-XL, ERCC1, Keratin 15,CD81/TAPA-1, CD9, Epithelial,Specific Antigen (ESA), and Mast CellChymase can also be provided. A plurality of probes for detecting one ormore miRNAs of an exosome can comprise probes for detecting one or moreof the following miRNAs: miR-9, miR-629, miR-141, miR-671-3p, miR-491,miR-182, miR-125a-3p, miR-324-5p, miR-148b, and miR-222,

The probes may be attached to a solid substrate, such as an array orbead. Alternatively, the probes are not attached. The detection systemmay be an array based system, a sequencing system, a PCR-based system,or a bead-based system, such as described above. For example, thedetection system can be a microfluidic device as described above.

The detection system may be part of a kit. Alternatively, the kit maycomprise the one or more probe sets, or plurality of probes, asdescribed herein. The kit may comprise probes for detecting an isolatedexosome, a plurality of exosomes, such as exosomes in a heterogeneouspopulation. The kit may comprise probes for detecting a homogeneouspopulation of exosomes. For example, the kit may comprise probes fordetecting a population of specific cell-of-origin exosomes, or exosomeswith the same specific bio-signature.

Portfolios

Portfolios of multiplexed markers to guide clinical decisions anddisease detection and management can be established such that thecombination of bio-signatures in the portfolio exhibit improvedsensitivity and specificity relative to individual bio-signatures orrandomly selected combinations of bio-signatures. In the context of theinstant invention, the sensitivity of the portfolio can be reflected inthe fold differences exhibited by a bio-signature's expression in thediseased state relative to the normal state. Specificity can bereflected in statistical measurements of the correlation of thesignaling of gene expression, for example, with the condition ofinterest (e.g. standard deviation can be a used as such a measurement).In considering a group of bio-signature for inclusion in a portfolio, asmall standard deviation in measurements correlates with greaterspecificity. Other measurements of variation such as correlationcoefficients can also be used in this capacity.

When combining biomakers or bio-signatures in this invention In VitroDiagnostic Multivariate Index Assays (IVDMIAs) guidelines andregulations may apply. IVDMIAs can apply to bio-signatures as defined asa set of 2 or more markers composed of any combination of genes, genealterations, mutations, amplifications, deletions, polymorphisms ormethylations, or proteins, peptides, polypeptides or RNA molecules,miRNAs, mRNAs, snoRNAs, hnRNAs or RNA that can be grouped so thatinformation obtained about the set of bio-signatures in the groupprovides a sound basis for making a clinically relevant judgment such asa diagnosis, prognosis, or treatment choice. These sets ofbio-signatures make up various portfolios of the invention. As with mostdiagnostic markers, it is often desirable to use the fewest number ofmarkers sufficient to make a correct medical judgment. This prevents adelay in treatment pending further analysis as well inappropriate use oftime and resources. Preferably, portfolios are established such that thecombination of bio-signatures in the portfolio exhibit improvedsensitivity and specificity relative to individual bio-signatures orrandomly selected combinations of bio-signatures. In the context of theinstant invention, the sensitivity of the portfolio can be reflected inthe fold differences exhibited by a bio-signature's expression in thediseased state relative to the normal state. Specificity can bereflected in statistical measurements of the correlation of thesignaling of gene expression, for example, with the condition ofinterest. In considering a group of markers in a bio-signature forinclusion in a portfolio, standard deviations, variances, co-variances,correlation coefficients, weighted averages, arithmetic sums, means,multiplicative values, weighted or balanced values or any mathematicalmanipulation of the values of 2 or more markers that can together beused to calculate a value or score that taken as a whole can be shown toproduce greater sensitivity, specificity, negative predictive value,positive predictive value or accuracy can also be used in this capacityand are within the scope of this invention.

In another embodiment pattern recognition methods can be used. Oneexample involves comparing biomarker expression profiles for variousbiomarkers (or bio-signature portfolios) to ascribe diagnoses. Theexpression profiles of each of the biomarker comprising thebio-signature portfolio are fixed in a medium such as a computerreadable medium.

In one example, a table can be established into which the range ofsignals (e.g., intensity measurements) indicative of disease orphysiological state is input. Actual patient data can then be comparedto the values in the table to determine whether the patient samples arenormal, benign, diseased, or represent a specific physiological state.In a more sophisticated embodiment, patterns of the expression signals(e.g., fluorescent intensity) are recorded digitally or graphically. Inthe example of RNA expression patterns from the biomarker portfoliosused in conjunction with patient samples are then compared to theexpression patterns. Pattern comparison software can then be used todetermine whether the patient samples have a pattern indicative of thedisease, a given prognosis, a pattern that indicates likeliness torespond to therapy, or a pattern that is indicative of a particularphysiological state. The expression profiles of the samples are thencompared to the portfolio of a control cell. If the sample expressionpatterns are consistent with the expression pattern(s) for disease,prognosis, or therapy-related response then (in the absence ofcountervailing medical considerations) the patient is diagnosed asmeeting the conditions that relate to these various circumstances. Ifthe sample expression patterns are consistent with the expressionpattern derived from the normal/control exosome population then thepatient is diagnosed negative for these conditions.

In another exemplary embodiment, a method for establishing biomarkerexpression portfolios is through the use of optimization algorithms suchas the mean variance algorithm widely used in establishing stockportfolios. This method is described in detail in the U.S. ApplicationPublication No. 20030194734, incorporated herein by reference.Alternatively, measured DNA alterations, changes in mRNA, protein, ormetabolites to phenotypic readouts of efficacy and toxicity may bemodeled and analyzed using algorithms, systems and methods described inU.S. Pat. Nos. 7,089,168, 7,415,359 and U.S. Application PublicationNos. 20080208784, 20040243354, or 20040088116, each of which is hereinincorporated by reference in its entirety.

An exemplary process of bio-signature portfolio selection andcharacterization of an unknown is summarized as follows:

1. Choose baseline class.

2. Calculate mean, and standard deviation of each biomarker for baselineclass samples.

3. Calculate (X*Standard Deviation+Mean) for each biomarker. This is thebaseline reading from which all other samples will be compared. X is astringency variable with higher values of X being more stringent thanlower.

4. Calculate ratio between each Experimental sample versus baselinereading calculated in step 3.

5. Transform ratios such that ratios less than 1 are negative (eg. usingLog base 10). (Under expressed biomarkers now correctly have negativevalues necessary for MV optimization).

6. These transformed ratios are used as inputs in place of the assetreturns that are normally used in the software application.

7. The software will plot the efficient frontier and return an optimizedportfolio at any point along the efficient frontier.

8. Choose a desired return or variance on the efficient frontier.

9. Calculate the Portfolio's Value for each sample by summing themultiples of each gene's intensity value by the weight generated by theportfolio selection algorithm.

10. Calculate a boundary value by adding the mean Bio-signaturePortfolio Value for Baseline groups to the multiple of Y and theStandard Deviation of the Baseline's Bio-signature Portfolio Values.Values greater than this boundary value shall be classified as theExperimental Class.

11. Optionally one can reiterate this process until best prediction.

The process of selecting a bio-signature portfolio can also include theapplication of heuristic rules. Preferably, such rules are formulatedbased on biology and an understanding of the technology used to produceclinical results. More preferably, they are applied to output from theoptimization method. For example, the mean variance method ofbio-signature portfolio selection can be applied to microarray data fora number of biomarkers differentially expressed in subjects with aspecific disease. Output from the method would be an optimized set ofbiomarkers that could include those that are expressed in exosomes aswell as in diseased tissue. If samples used in the testing method areobtained from exosomes and certain biomarkers differentially expressedin instances of disease or physiological state could also bedifferentially expressed in exosomes, then a heuristic rule can beapplied in which a bio-signature portfolio is selected from theefficient frontier excluding those that are differentially expressed inexosomes. Of course, the rule can be applied prior to the formation ofthe efficient frontier by, for example, applying the rule during datapre-selection.

Other statistical, mathematical and computational algorithms for theanalysis of linear and non-linear feature subspaces, feature extractionand signal deconvolution in large scale datasets to identifyexosome-derived multiplex analyte profiles for diagnosis, prognosis andtherapy selection and/or characterization of define physiological statescan be done using any combination of unsupervised analysis methods,including but not limited to: principal component analysis (PCA) andlinear and non-linear independent component analysis (ICA); blind sourceseparation, nongaussinity analysis, natural gradient maximum likelihoodestimation; joint-approximate diagonalization; eigenmatrices; Gaussianradical basis function, kernel and polynominal kernel analysissequential floating forward selection.

Computer Systems

An exosome can be assayed for molecular features, for example, bydetermining an amount, presence or absence of one or more biomarkerssuch as listed FIGS. 1, 3-60. The data generated can be used to producea bio-signature, which can be stored and analyzed by a computer system,such as shown in FIG. 62. The assaying or correlating of thebio-signature with one or more phenotypes can also be performed bycomputer systems, such as by using computer executable logic.

A computer system, such as shown in FIG. 62, can be used to transmitdata and results following analysis. Accordingly, FIG. 62 is a blockdiagram showing a representative example logic device through whichresults from exosome analysis can be reported or generated. FIG. 62shows a computer system (or digital device) 800 to receive and storedata generated from exosome analysis, analyze the data to generate oneor more bio-signatures, and produce a report of the one or morebio-signatures. The computer system can also perform comparisons andanalyses of bio-signatures generated, and transmit the results.Alternatively, the computer system can receive raw data of exosomeanalysis, such as through transmission of the data over a network, andperform the analysis.

The computer system 800 may be understood as a logical apparatus thatcan read instructions from media 811 and/or network port 805, which canoptionally be connected to server 809 having fixed media 812. The systemshown in FIG. 62 includes CPU 801, disk drives 803, optional inputdevices such as keyboard 815 and/or mouse 816 and optional monitor 807.Data communication can be achieved through the indicated communicationmedium to a server 809 at a local or a remote location. Thecommunication medium can include any means of transmitting and/orreceiving data. For example, the communication medium can be a networkconnection, a wireless connection or an Internet connection. Such aconnection can provide for communication over the World Wide Web. It isenvisioned that data relating to the present invention can betransmitted over such networks or connections for reception and/orreview by a party 822. The receiving party 822 can be but is not limitedto an individual, a health care provider or a health care manager. Inone embodiment, a computer-readable medium includes a medium suitablefor transmission of a result of an analysis of a biological sample, suchas exosome bio-signatures. The medium can include a result regarding anexosome bio-signature of a subject, wherein such a result is derivedusing the methods described herein.

Ex vivo Harvesting of Exosomes

Exosomes for analysis and determination of a phenotype can also be fromex vivo harvesting. Cells can be cultured and that exosomes releasedfrom cells of interest in culture either result spontaneously or can bestimulated to release exosomes into the medium. (see for example,Zitvogel, et al./998. Nat. Med. 4: 594-600; Chaput, et al. 2004. J.Immunol. 172: 2137-214631: 2892-2900; Escudier, et al. 2005. J. Transl.Med. 3: 10; Morse, et al. 2005, J. Transl. Med. 3: 9; Peche, et al.2006. Am. J. Transplant. 6: 1541-1550; Kim, et al. 2005. J. Immunol.174: 6440-6448, all of which are herein incorporated by reference intheir entireties). Cell lines or tissue samples can be grown to 80%confluence before being cultured in fresh DMEM for 72 h. Subsequentexosome production can be stimulated (see, for example, heat shocktreatment of melanoma cells as described by Dressel, et al. 2003. CancerRes. 63: 8212-8220, which is herein incorporated by reference in itsentirety). The supernatant can then be harvested and exosomes preparedas described herein.

Exosomes produced ex vivo can, in one example, be cultured from acell-of-origin or cell line of interest, exosomes can be isolated fromthe cell culture medium and subsequently labeled with a magnetic label,a fluorescent moiety, a radioisotope, an enzyme, a chemiluminescentprobe, a metal particle, a non-metal colloidal particle, a polymeric dyeparticle, a pigment molecule, a pigment particle, an electrochemicallyactive species, semiconductor nanocrystal or other nanoparticlesincluding quantum dots or gold particles to be reintroduced in vivo as alabel for imaging analysis. Ex vivo cultured exosomes can alternativelybe used to identify novel bio-signatures by setting up culturingconditions for a given cell-of-origin with characteristics of interest,for example a culture of lung cancer cells or cell line with a knownEGFR mutation that confers resistant to or susceptibility to gefitinib,then exposing the cell culture to gefitinib, isolating exosomes thatarise from the culture and subsequently analyzing them on a discoveryarray to look for novel antigens or binding agents expressed on theoutside of exosomes that could be used as a bio-signature to capturethis species of exosome. Additionally, it would be possible to isolateany other biomarkers or bio-signatures found within these exosomes fordiscovery of novel signatures (including but not limited to nucleicacids, proteins, lipids, or combinations thereof) that may have clinicaldiagnostic, prognostic or therapy related implication.

Cells of interest can also be first isolated and cultured from tissuesof interest. For example, human hair follicles in the growing phase,anagen, can be plucked individually from a patient's scalp using sterileequipment and plasticware, taking care not to damage the follicle. Eachsample can be transferred to a Petri dish containing sterile PBS fortissue culture. Isolated human anagen hair follicles can be carefullytransferred to an individual well of a 24-well plate containing 1 ml ofWilliam's E medium. Follicles can be maintained free-floating at 37° C.in an atmosphere of 5% CO₂ and 95% air in a humidified incubator. Mediumcan be changed every 3 days, taking care not to damage the follicles.Cells can then be collected and spun down from the media. Exosomes maythen be isolated using antigens or cellular binding partners that arespecific to such cell-of-origin specific exosomes using methods aspreviously described. Biomarkers and bio-signatures can then be isolatedand characterized by methods known to those skilled in the art.

Cells of interest may also be cultured under microgravity orzero-gravity conditions or under a free-fall environment. For example,NASA's bioreactor technology will allow such cells to be grown at muchfaster rate and in much greater quantities. Exosomes may then beisolated using antigens or cellular binding partners that are specificto such cell-of-origin specific exosomes using methods as previouslydescribed.

Rotating wall vessels or RWVersus are a class of bioreactors developedby and for NASA that are designed to grow suspension cultures of cellsin a quiescent environment that simulates microgravity can also be used.(see for example, U.S. Pat. Nos. 5,026,650; 5,153,131; 5,153,133;5,437,998; 5,665,594; 5,702,941; 7,351,584, 5,523,228, 5,104,802,6,117,674, Schwarz, R P, et al., J. Tiss. Cult. Meth. 14:51-58, 1992;Martin et al., Trends in biotechnology 2004; 22; 80-86, Li et al.,Biochemical Engineering Journal 2004; 18; 97-104, Ashammakhi et al.,Journal Nanoscience Nanotechnology 2006; 9-10: 2693-2711, Zhang et al.,International Journal of Medicine 2007; 4: 623-638, Cowger, N L, et al.,Biotechnol. Bioeng 64:14-26, 1999, Spaulding, G F, et al., J. Cell.Biochem. 51:249-251, 1993, Goodwin, T J, et al., Proc. Soc. Exp. Biol.Med. 202:181-192, 1993; Freed, L E et al., In Vitro Cell. Dev. Biol.33:381-385, 1997, Clejan, S. et al, Biotechnol. Bioeng. 50:587-597,1996). Khaoustov, V I, et al., In Vitro Cell. Dev. Biol. 35:501-509.1999, each of which is herein incorporated by reference in itsentirety).

Alternatively, cells of interest or cell-of-origin specific exosomesthat have been isolated may be cultured in a stationary phase plug-flowbioreactor as generally described in U.S. Pat. No. 6,911,201, and U.S.Application Publication Nos. 20050181504, 20050180958, 20050176143 and20050176137, each of which is herein incorporated by reference in itsentirety. Alternatively, cells of interest or cell-origin specificexosomes may also be isolated and cultured as generally described inU.S. Pat. No. 5,486,359.

One embodiment can include the steps of providing a tissue specimencontaining cells of interest or cell-origin specific exosomes, addingcells or exosomes from the tissue specimen to a medium which allows,when cultured, for the selective adherence of only the cells of interestor cell-origin specific exosomes to a substrate surface, culturing thespecimen-medium mixture, and removing the non-adherent matter from thesubstrate surface is generally described in U.S. Pat. No. 5,486,359,which is herein incorporated by reference in its entirety.

Exosomes as Imaging Tools

In other embodiments, exosomes can be used as imaging tools. Labeledcirculating tumor cells (CTCs) can be noninvasively visualized in vivoas they flow through the peripheral vasculature (He, W et al. (2007)PNAS 104(28)11760-11765). The method can involve i.v. injection of atumor-specific fluorescent ligand followed by multiphoton fluorescenceimaging of superficial blood vessels to quantitate the flowing CTCs.Studies in mice with metastatic tumors demonstrated that CTCs can bequantitated weeks before metastatic disease is detected by other means.Similar methods could be used and applied to circulating cell-of-originspecific exosomes as well. The decision to administer chemotherapy aftertumor resection usually depends on an oncologist's assessment of thepresence of microscopic metastatic disease. Although computedtomography, MRI, tissue/sentinel lymph node biopsy or serum cancermarker analysis can each detect some level of residual disease, thepresence of circulating tumor derived exosomes can correlate mostsensitively with cancer progression and metastasis. Noninvasive imagingof these exosomes in real time as they flow through the peripheralvasculature could improve detection sensitivity by enabling analysis ofsignificantly larger blood volumes (potentially the entire blood volumeof the patient). Exosomes isolated from bodily fluid, purified, labeledand then reintroduced into the system can be used for identification ofearly tumors not yet visible by traditional imaging methods (e.g. earlybreast tumors or early ovarian tumor cells). Labeled exosomes can alsobe used as a signal to identify tumors of metastatic potential.

In one embodiment, exosomes can be labeled by peptide/antigen targetingto label the exosomes either in vivo or in vitro and then reintroduce inthe circulatory system for the purposes of diagnostic imaging. Suitablelabels may include those that may be detected by intravital flowcytometry, X-radiography, NMR, PET/SPECT or MRI. For X-radiographictechniques, suitable labels include any radioisotope that emitsdetectable radiation but that is not overtly harmful to the patient,such as barium or cesium, for example. Suitable labels for NMR or MRIgenerally include those with a detectable characteristic spin, such asdeuterium. Suitable imaging systems may be used to detect the labeledexosomes in the circulatory system.

The labeled exosomes can be administered by arterial or venousinjection, and can be formulated as a sterile, pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable solutions, having due regard to pH, isotonicity,stability, and the like, is within the skill in the art. A preferredformulation for intravenous injection should contain, in addition to thelabeled exosomes, an isotonic vehicle such as Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, Lactated Ringer's Injection, or other vehicle. An effectiveamount of labeled exosomes can be an amount sufficient to yield anacceptable image using equipment which is available for clinical use. Aneffective amount of the labeled exosomes may be administered in morethan one injection. Effective amounts of the labeled exosomes will varyaccording to factors such as the degree of susceptibility of theindividual, the age, sex, and weight of the individual, idiosyncraticresponses of the individual, the dosimetry. Effective amounts of thelabeled exosomes will also vary according to instrument and film-relatedfactors.

In a further embodiment, intravital flow cytometry can be used tononinvasively count labeled exosomes in vivo as they flow through theperipheral vasculature. The method can include i.v. injection of atumor-specific fluorescent ligand followed by multiphoton fluorescenceimaging of superficial blood vessels to quantitate the flowing exosomes.Intravital flow cytometry for detection of exosomes circumvents samplinglimitations and renders quantitation of rare events statisticallysignificant by enabling analysis of the majority of a patient's bloodvolume (≈5 liters).

Many human carcinomas overexpress a receptor for the vitamin folic acid(>90% of ovarian and endometrial cancers, 86% of kidney cancers, 78% ofnonsmall cell lung cancers, etc). Alternatively, normal tissues eitherlack measurable folate receptors (FR) or express FR at a site that isinaccessible to parenterally administered drugs. Because FR-expressingcancer masses can be selectively labeled in vivo by injection of eitherradioactive or fluorescent folate conjugates that bind FR with nanomolaraffinity it is possible for single exosomes to bind sufficient numbersof folate conjugates to allow their detection in vivo as they passthrough a patient's peripheral vasculature. To increasesignal-to-background ratios, a tumor-specific probe is used that rapidlyclear circulation if left uncaptured by exosomes. For this purpose,folate-dye conjugates (e.g. folate-AlexaFluor 488) conjugates can beused because tumor-specific antibodies were found to promote phagocyticclearance of the exosomes to which they bound, thereby causingsignificant underestimation of exosomes counts. To further ensure thatthe cells labeled with folate-AlexaFluor 488 are indeed malignant,monoclonal anti-human antibodies can be used (e.g. CA125 for ovariancancer) plus an appropriate secondary antibody conjugated torhodamine-X.

In another embodiment, exosomes can be labeled in vivo by intravenouslyintroducing a labeling agent that specifically targets the exosome fordownstream imaging applications similar to those described above.

Reimbursement Codes

In one embodiment, the use of exosomes as diagnostic, therapy-related orprognostic markers in the identification of disease, disease stage,progression or therapy can be assigned specific U.S. Medicarereimbursement codes. In one embodiment, the isolation and the use ofcell-of-origin specific exosomes are used. The reimbursement code may bea code developed under the National Council for Prescription DrugPrograms Professional Pharmacy Services (NCPDP/PPS code). Areimbursement code can be a diagnosis code utilized or recognized by aninsurance company, for example. The diagnostic code is assignable basedupon a reimbursement requirement by a third party. Alternatively, thediagnostic code is assignable based upon a need to analyze theutilization of medical resources. A set of diagnosis codes can conformto and/or be compatible with, for example, ICD (InternationalClassification of Diseases) codes, 9th Edition, Clinical Modification,(ICD-9-CM), Volumes 1, 2 and 3; ICD-10, which is maintained anddistributed by the U.S. Health and Human Services department; HCPCS(Health Care Financing Administration Common Procedure Coding System);NDC (National Drug Codes); CPT-4 (Current Procedural Terminology);Fourth Edition CDPN (Code on Dental Procedures and Nomenclature);SNOMED-RT “Systematicized Nomenclature of Medicine, ReferenceTerminology” by the College of American Pathologists; UMLS (UnifiedMedical Language System), by the National Library of Medicine; LOINCLogical Observation Identifiers, Names, and Codes; Regenstrief Instituteand the Logical Observation Identifiers Names and Codes (LOINC®)Committee; Clinical Terms also known as “Read Codes”; DIN DrugIdentification Numbers; Reimbursement Classifications including DRGs(Diagnosis Related Groups); CDT Current Dental Terminology; NIC (Nursingintervention codes); or Commercial Vocabulary Services (such asHealthLanguage by HealthLanguage Inc.), each of which is incorporated byreference in its entirety.

In one embodiment, each of the isolation methods for exosomes describedherein can be assigned a specific reimbursement code. For example, eachof the isolation methods of cell-of-origin specific exosomes describedherein can be assigned a specific reimbursement code. In anotherembodiment, the specific bio-signature(s) obtained from the analysis ofexosomes can be assigned a specific reimbursement code. In yet anotherembodiment, the specific bio-signature(s) obtained from the analysis ofcell-of-origin specific exosomes can be assigned a specificreimbursement code. Alternatively, kits for the detection of aparticular bio-signature of exosomes in a biological sample can beassigned to a specific reimbursement code. Alternatively, kits for thedetection of a particular bio-signature of specific cell-of-originexosomes in a biological sample can be assigned to a specificreimbursement code.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1 Purification of Exosomes from Prostate Cancer CellLines

Prostate cancer cell lines are cultured for 3-4 days in culture mediacontaining 20% FBS (fetal bovine serum) and 1% P/S/G. The cells are thenpre-spun for 10 minutes at 400×g at 4° C. The supernatant is kept andcentrifuged for 20 minutes at 2000×g at 4. The supernatant containingexosomes can be concentrated using a Millipore Centricon Plus-70 (Cat #UFC710008 Fisher).

The Centricon is pre washed with 30 mls of PBS at 1000×g for 3 minutesat room temperature. Next, 15-70 mls of the pre-spun cell culturesupernatant is poured into the Concentrate Cup and is centrifuged in aSwing Bucket Adapter (Fisher Cat #75-008-144) for 30 minutes at 1000×gat room temperature.

The flow through in the Collection Cup is poured off. The volume in theConcentrate Cup is brought back up to 60 mls with any additionalsupernatant. The Concentrate Cup is centrifuged for 30 minutes at 1000×gat room temperature until all of the cell supernatant is concentrated.

The Concentrate Cup is washed by adding 70 m-1s of PBS and centrifugedfor 30-60 minutes at 1000×g until approximately 2 mls remains. Theexosomes are removed from the filter by inverting the concentrate intothe small sample cup and centrifuge for 1 minute at 4° C. The volume isbrought up to 25 mls with PBS. The exosomes are now concentrated and areadded to a 30% Sucrose Cushion.

To make a cushion, 4 mls of Tris/30% Sucrose/D2O solution (30 gprotease-free sucrose, 2.4 g Tris base, 50 ml D2O, adjust pH to 7.4 withION NCL drops, adjust volume to 100 mls with D2O, sterilize by passingthru a 0.22-um filter) is loaded to the bottom of a 30 ml V bottom thinwalled Ultracentrifuge tube. The diluted 25 mls of concentrated exosomesis gently added above the sucrose cushion without disturbing theinterface and is centrifuged for 75 minutes at 100,000×g at 4° C. The˜25 mls above the sucrose cushion is carefully removed with a 10 mlpipet and the ˜3.5 mls of exosome is collected with a fine tip transferpipet (SAMCO 233) and transferred to a fresh ultracentrifuge tube, where30 mls PBS is added. The tube is centrifuged for 70 minutes at 100,000×gat 4° C. The supernatant is poured off carefully. The pellet isresuspended in 200 ul PBS and can be stored at 4° C. or used for assays.A BCA assay (1:2) can be used to determine protein content and Westernblotting or electron micrography can be used to determine exosomepurification.

Example 2 Purification of Exosomes from VCaP and 22Rv1

Exosomes from Vertebral-Cancer of the Prostate (VCaP) and 22Rv1, a humanprostate carcinoma cell line, derived from a human prostatic carcinomaxenograft (CWR22R) were collected by ultracentrifugation by firstdiluting plasma with an equal volume of PBS (1 ml). The diluted fluidwas transferred to a 15 ml falcon tube and centrifuged 30 minutes at2000×g 4° C. The supernatant (˜2 mls) was transferred to anultracentrifuge tube 5.0 ml PA thinwall tube (Sorvall #03127) andcentrifuged at 12,000×g, 4° C. for 45 minutes.

The supernatant (˜2 mls) was transferred to a new 5.0 ml ultracentrifugetubes and filled to maximum volume with addition of 2.5 mls PBS andcentrifuged for 90 minutes at 110,000×g, 4° C. The supernatant waspoured off without disturbing the pellet and the pellet resuspended with1 ml PBS. The tube was filled to maximum volume with addition of 4.5 mlof PBS and centrifuged at 110,000×g, 4° C. for 70 minutes.

The supernatant was poured off without disturbing the pellet and anadditional 1 ml of PBS was added to wash the pellet. The volume wasincreased to maximum volume with the addition of 4.5 mls of PBS andcentrifuged at 110,000×g for 70 minutes at 4° C. The supernatant wasremoved with P-1000 pipette until ˜100 μl of PBS was in the bottom ofthe tube. The ˜90 μl remaining was removed with P-200 pipette and thepellet collected with the ˜10 μl of PBS remaining by gently pipettingusing a P-20 pipette into the microcentrifuge tube. The residual pelletwas washed from the bottom of a dry tube with an additional 5 μl offresh PBS and collected into microcentrifuge tube and suspended inphosphate buffered saline (PBS) to a concentration of 500 μg/ml.

Example 3 Plasma Collection and Exosome Purification

Blood is collected via standard veinpuncture in a 7 ml K2-EDTA tube. Thesample is spun at 400 g for 10 minutes in a 4° C. centrifuge to separateplasma from blood cells (SORVALL Legend RT+ centrifuge). The supernatant(plasma) is transferred by careful pipetting to 15 ml Falcon centrifugetubes. The plasma is spun at 2,000 g for 20 minutes and the supernatantis collected.

For storage, approximately 1 ml of the plasma (supernatant) is aliquotedto a cryovials, placed in dry ice to freeze them and stored in −80° C.Before exosome purification, if samples were stored at −80° C., samplesare thawed in a cold water bath for 5 minutes. The samples are mixed endover end by hand to dissipate insoluble material.

In a first prespin, the plasma is diluted with an equal volume of PBS(example, approximately 2 ml of plasma is diluted with 2 ml of PBS). Thediluted fluid is transferred to a 15 ml Falcon tube and centrifuged for30 minutes at 2000×g at 4° C.

For a second prespin, the supernatant (approximately 4 mls) is carefullytransferred to a 50 ml Falcon tube and centrifuged at 12,000×g at 4° C.for 45 minutes in a Sorval.

In the isolation step, the supernatant (approximately 2 mls) iscarefully transferred to a 5.0 ml ultracentrifuge PA thinwall tube(Sorvall #03127) using a P1000 pipette and filled to maximum volume withan additional 0.5 mls of PBS. The tube is centrifuged for 90 minutes at110,000×g at 4° C.

In the first wash, the supernatant is poured off without disturbing thepellet. The pellet is resuspended or washed with 1 ml PBS and the tubeis filled to maximum volume with an additional 4.5 ml of PBS. The tubeis centrifuged at 110,000×g at 4° C. for 70 minutes. A second wash isperformed by repeating the same steps.

The exosomes are collected by removing the supernatant with P-1000pipette until approximately 100 μl of PBS is in the bottom of the tube.Approximately 90 μl 1 of the PBS is removed and discarded with P-200pipette. The pellet and remaining PBS is collected by gentle pipettingusing a P-20 pipette. The residual pellet is washed from the bottom ofthe dry tube with an additional 5 μl of fresh PBS and collected into amicrocentrifuge tube.

Example 4 Analysis of Exosomes Using Antibody-Coupled Microspheres andDirectly Conjugated Antibodies

This example demonstrates the use of particles coupled to an antibody,where the antibody captures the exosomes (see for example, FIG. 64A). Anantibody, the detector antibody, is directly coupled to a label, and isused to detect a biomarker on the captured exosome.

First, an antibody-coupled microsphere set is selected (Luminex, Austin,Tex.). The microsphere set can comprise various antibodies, and thusallows multiplexing. The microspheres are resuspended by vortex andsonication for approximately 20 seconds. A Working Microsphere Mixtureis prepared by diluting the coupled microsphere stocks to a finalconcentration of 100 microspheres of each set/μL in Startblock (Pierce(37538)). (Note: 50 μL of Working Microsphere Mixture is required foreach well.) Either PBS-1% BSA or PBS-BN(PBS, 1% BSA, 0.05% Azide, pH7.4) may be used as Assay Buffer.

A 1.2 μm Millipore filter plate is pre-wet with 100 μl/well of PBS-1%BSA (Sigma (P3688-10PAK+0.05% NaAzide (S8032))) and aspirated by vacuummanifold. An aliquot of 50 μl of the Working Microsphere Mixture isdispensed into the appropriate wells of the filter plate (MilliporeMultiscreen HTS (MSBVN1250)). A 50 μl aliquot of standard or sample isdispensed into to the appropriate wells. The filter plate is covered andincubated for 60 minutes at room temperature on a plate shaker. Theplate is covered with a sealer, placed on the orbital shaker and set to900 for 15-30 seconds to re-suspend the beads. Following that the speedis set to 550 for the duration of the incubation.

The supernatant is aspirated by vacuum manifold (less than 5 inches Hgin all aspiration steps). Each well is washed twice with 100 μl ofPBS-1% BSA (Sigma (P3688-10PAK+0.05% NaAzide (S8032))) and is aspiratedby vacuum manifold. The microspheres are resuspended in 50 μL of PBS-1%BSA (Sigma (P3688-10PAK+0.05% NaAzide (S8032))). The PE conjugateddetection antibody is diluted to 4 μg/mL (or appropriate concentration)in PBS-1% BSA (Sigma (P3688-10PAK+0.05% NaAzide (S8032))). (Note: 50 μLof diluted detection antibody is required for each reaction.) A 50 μlaliquot of the diluted detection antibody is added to each well. Thefilter plate is covered and incubated for 60 minutes at room temperatureon a plate shaker. The filter plate is covered with a sealer, placed onthe orbital shaker and set to 900 for 15-30 seconds to re-suspend thebeads. Following that the speed is set to 550 for the duration of theincubation. The supernatant is aspirated by vacuum manifold. The wellsare washed twice with 100 μl of PBS-1% BSA (Sigma (P3688-10PAK+0.05%NaAzide (S8032))) and aspirated by vacuum manifold. The microspheres areresuspended in 100 μl of PBS-1′% BSA (Sigma (P3688-10PAK+0.05% NaAzide(S8032))). The microspheres are analyzed on a Luminex analyzer accordingto the system manual.

Example 5 Analysis of Exosomes Using Antibody-Coupled Microspheres andBiotinylated Antibody

This example demonstrates the use of particles coupled to an antibody,where the antibody captures the exosomes. An antibody, the detectorantibody, is biotinylated. A label coupled to streptavidin is used todetect the biomarker.

First, the appropriate antibody-coupled microsphere set is selected(Luminex, Austin, Tex.). The microspheres are resuspended by vortex andsonication for approximately 20 seconds. A Working Microsphere Mixtureis prepared by diluting the coupled microsphere stocks to a finalconcentration of 50 microspheres of each set/μL in Startblock (Pierce(37538)). (Note: 50 μl of Working Microsphere Mixture is required foreach well.) Beads in Start Block should be blocked for 30 minutes and nomore than 1 hour.

A 1.2 μm Millipore filter plate is pre-wet with 100 μl/well of PBS-1%BSA+Azide (PBS-BN)((Sigma (P3688-10PAK+0.05% NaAzide (S8032))) and isaspirated by vacuum manifold. A 50 μl aliquot of the Working MicrosphereMixture is dispensed into the appropriate wells of the filter plate(Millipore Multiscreen HTS (MSBVN1250)). A 50 μl aliquot of standard orsample is dispensed to the appropriate wells. The filter plate iscovered with a seal and is incubated for 60 minutes at room temperatureon a plate shaker. The covered filter plate is placed on the orbitalshaker and set to 900 for 15-30 seconds to re-suspend the beads.Following that, the speed is set to 550 for the duration of theincubation.

The supernatant is aspirated by a vacuum manifold (less than 5 inches Hgin all aspiration steps). Aspiration can be done with the Pall vacuummanifold. The valve is place in the full off position when the plate isplaced on the manifold. To aspirate slowly, the valve is opened to drawthe fluid from the wells, which takes approximately 3 seconds for the100 μl of sample and beads to be fully aspirated from the well. Once allof the sample is drained, the purge button on the manifold is pressed torelease residual vacuum pressure from the plate.

Each well is washed twice with 100 μl of PBS-1% BSA+Azide (PBS-BN)(Sigma(P3688-10PAK+0.05% NaAzide (S8032))) and is aspirates by vacuummanifold. The microspheres are resuspended in 50 μl of PBS-1% BSA+Azide(PBS-BN)((Sigma (P3688-10PAK+0.05% NaAzide (S8032)))

The biotinylated detection antibody is diluted to 4 μg/mL in PBS-1%BSA+Azide (PBS-BN)((Sigma (P3688-10PAK+0.05% NaAzide (S8032))). (Note:50 μl of diluted detection antibody is required for each reaction.) A 50μl aliquot of the diluted detection antibody is added to each well.

The filter plate is covered with a sealer and is incubated for 60minutes at room temperature on a plate shaker. The plate is placed onthe orbital shaker and set to 900 for 15-30 seconds to re-suspend thebeads. Following that, the speed is set to 550 for the duration of theincubation.

The supernatant is aspirated by vacuum manifold. Aspiration can be donewith the Pall vacuum manifold. The valve is place in the full offposition when the plate is placed on the manifold. To aspirate slowly,the valve is opened to draw the fluid from the wells, which takesapproximately 3 seconds for the 100 ul of sample and beads to be fullyaspirated from the well. Once all of the sample is drained, the purgebutton on the manifold is pressed to release residual vacuum pressurefrom the plate.

Each well is washed twice with 100 μl of PBS-1% BSA+Azide(PBS-BN)((Sigma (P3688-10PAK+0.05% NaAzide (S8032))) and is aspirated byvacuum manifold. The microspheres are resuspended in 50 μl of PBS-1% BSA(Sigma (P3688-10PAK+0.05% NaAzide (S8032))).

The streptavidin-R-phycoerythrin reporter (Molecular Probes 1 mg/ml) isdiluted to 4 μg/mL in PBS-1% BSA+Azide (PBS-BN)(. (Note: 50 μl ofdiluted streptavidin-R-phycoerythrin is required for each reaction.) A50 μl aliquot of the diluted streptavidin-R-phycoerythrin is added toeach well.

The filter plate is covered with a sealer and is incubated for 60minutes at room temperature on a plate shaker. The plate is placed onthe orbital shaker and set to 900 for 15-30 seconds to re-suspend thebeads. Following that, the speed is set to 550 for the duration of theincubation.

The supernatant is aspirated by vacuum manifold. Aspiration can be donewith the Pall vacuum manifold. The valve is place in the full offposition when the plate is placed on the manifold. To aspirate slowly,the valve is opened to draw the fluid from the wells, which takesapproximately 3 seconds for the 100 ul of sample and beads to be fullyaspirated from the well. Once all of the sample is drained, the purgebutton on the manifold is pressed to release residual vacuum pressurefrom the plate.

Each well is washed twice with 100 μl of PBS-1% BSA+Azide(PBS-BN)((Sigma (P3688-10PAK+0.05% NaAzide (S8032))) and is aspirated byvacuum manifold. The microspheres are resuspended in 100 μl of PBS-1%BSA+Azide (PBS-BN)((Sigma (P3688-10PAK+0.05% NaAzide (S8032))) andanalyzed on the Luminex analyzer according to the system manual.

Example 6 Determining Bio-Signatures for Prostate Cancer UsingMultiplexing

The exosomes samples obtained using methods as described in Example 1-3are used in multiplexing assays as described in Examples 4 and 5. Thedetection antibodies used are CD63, CD9, CD81, B7H3 and EpCam. Thecapture antibodies used are CD9, PSCA, TNFR, CD63 2X, B7H3, MFG-E8,EpCam 2×, CD63, Rab, CD81, SETAP, PCSA, PSMA, 5T4, Rab IgG (control) andIgG (control), resulting in 100 combinations to be screened (FIG. 64B).

Ten prostate cancer patients and 12 normal control patients werescreened. The results are depicted in FIG. 68 and FIG. 70A. FIG. 70Bdepicts the results of using PCSA capture antibodies (FIG. 70B, leftgraph) or EpCam capture antibodies (FIG. 70B, right graph), anddetection using one or more detector antibodies. The sensitivity andspecificity of the different combinations is depicted in FIG. 73.

Example 7 Determining Bio-Signatures for Colon Cancer Using Multiplexing

The exosomes samples obtained using methods as described in Example 3 isused in multiplexing assays as described in Examples 4 and 5. Thedetection antibodies used are CD63, CD9, CD81, B7H3 and EpCam. Thecapture antibodies used are CD9, PSCA, TNFR, CD63 2X, B7H3, MFG-E8,EpCam 2X, CD63, Rab, CD81, STEAP, PCSA, PSMA, 5T4, Rab IgG (control) andIgG (control), resulting in 100 combinations to be screened.

The results are depicted in FIGS. 69, 71, and 72. The sensitivity of thedifferent combinations is depicted in FIG. 74.

Example 9 Capture of Exosomes Using Magnetic Beads

Exosomes isolated as described in Example 2 are used. Approximately 40ul of the exosomes are incubated with approximately 5 ug (˜50 μl) ofEpCam antibody coated Dynal beads (Invitrogen, Carlsbad, Calif.) and 50μl of Starting Block. The exosomes and beads are incubated with shakingfor 2 hours at 45° C. in a shaking incubator. The tube containing theDynal beads is placed on the magnetic separator for 1 minute and thesupernatant removed. The beads are washed twice and the supernatantremoved each time. Wash beads twice, discarding the supernatant eachtime.

Example 10 Detection of TMPRSS2:ERG in Exosomes

The RNA from the bead-bound exosomes of Example 9 was isolated using theQiagen miRneasy™ kit, (Cat. No. 217061), according to the manufacturer'sinstructions.

The exosomes are homogenized in QIAzol™ Lysis Reagent (Cat. No. 79306).After addition of chloroform, the homogenate is separated into aqueousand organic phases by centrifugation. RNA partitions to the upper,aqueous phase, while DNA partitions to the interphase and proteins tothe lower, organic phase or the interphase. The upper, aqueous phase isextracted, and ethanol is added to provide appropriate bindingconditions for all RNA molecules from 18 nucleotides (nt) upwards. Thesample is then applied to the RNeasy™ Mini spin column, where the totalRNA binds to the membrane and phenol and other contaminants areefficiently washed away. High quality RNA is then eluted in RNase-freewater.

RNA from the VCAP bead captured exosomes was measured with the TaqmanTMPRSS:ERG fusion transcript assay (Kirsten D. Mertz et al. Neoplasia.2007 March; 9(3): 200-206.). RNA from the 22Rv1 bead captured exosomeswas measured with the Taqman SPINK1 transcript assay (Scott A. Tomlin etal. Cancer Cell 2008 June 13(6):519-528). The GAPDH transcript (controltranscript) was also measured for both sets of exosomal RNA.

Higher CT values indicate lower transcript expression. One change incycle threshold (CT) is equivalent to a 2 fold change, 3 CT differenceto a 4 fold change, and so forth, which can be calculated with thefollowing: 2̂^(CT1-CT2). This experiment shows a difference in CT of theexpression of the fusion transcript TMPRSS:ERG and the equivalentcaptured with the IgG2 negative control bead (FIG. 75). The samecomparison of the SPINK1 transcript in 22RV1 exosomes shows a CTdifference of 6.14 for a fold change of 70.5 (FIG. 75C).

Example 11 MicroRNA Profiles in Exosomes

Exosomes were collected by ultracentrifugation from 22Rv1, LNCaP, Vcapand normal plasma (pooled from 16 donors) as described in Examples 1 and2. RNA was extracted using the Exiqon miR isolation kit (Cat. No.300110, 300111). Equals amounts of exosomes (30 μg) were used asdetermined by BCA assay.

Equal volumes (5 μl) were put into a reverse-transcription reaction formicroRNA. The reverse-transcriptase reactions were diluted in 81 μl ofnuclease-free water and then 9 μl of this solution was added to eachindividual miR assay. MiR-629 was found to only be expressed in PCa(prostate cancer) exosomes and was virtually undetectable in normalplasma exosomes. MiR-9 was found to be highly overexpressed (˜704 foldincrease over normal as measured by copy number) in all PCa cell lines,and has very low expression in normal plasma exosomes. The top tendifferentially expressed miRNAs are depicted in FIG. 76.

Example 12 MicroRNA Profiles of Magentic EpCam-Captured Exosomes

The bead-bound exosomes of Example 9 was placed in QIAzol™ Lysis Reagent(Cat. #79306). An aliquot of 125 fmol of c. elegans miR-39 was added.The RNA from the exosomes was isolated using the Qiagen miRneasy™ kit,(Cat. #217061), according to the manufacturer's instructions, and elutedin 30 ul RNAse free water.

10 μl of the purified RNA was placed into a pre-amplification reactionfor miR-9, miR-141 and miR-629 using a Veriti 96-well thermocycler. A1:5 dilution of the pre-amplification solution was used to set up aqRT-PCR reaction for miR9 (ABI 4373285), miR-141 (ABI 4373137) andmiR-629 (ABI 4380969) as well as c. elegans miR-39 (ABI 4373455). Theresults were normalized to the c. elegans results for each sample.

Example 13 MicroRNA Profiles of CD9-Captured Exosomes

The CD9 coated Dynal beads (Invitrogen, Carlsbad, Calif.) were usedinstead of EpCam coated beads as in Example 12. Exosomes from prostatecancer patients, LNCaP, or normal purified exomes were incubated withthe CD9 coated beads and the RNA isolated as described in Example 12.The expression of miR-21 and miR-141 was detected by qRT-PCR and theresults depicted in FIGS. 77 and 78.

Example 14 Reference Values for Prostate Cancer

Fourteen stage 3 prostate cancer subjects, eleven benign prostatehyperplasia (BPH) samples, and 15 normal samples were tested. Exosomesamples were obtained using methods as described in Example 3 and usedin multiplexing assays, such as described in Examples 4 and 5. Thesamples were analyzed to determine four criteria 1) if the sample hasoverexpressed exosomes, 2) if the sample has overexpressed prostateexosomes, 3) if the sample has overexpressed cancer exosomes, and 4) ifthe sample is reliable. If the sample met all four criteria, thecategorization of the sample as positive for prostate cancer had varyingsensitivities and specificities, depending on the differentbio-signatures present for a sample as described below (Cancer-1,Cancer-2, and Cancer-3, FIG. 79). The four criteria were as follows:

Exosome Overexpression

The mean fluorescence intensities (MFIs) for a sample in three assayswere averaged to determine a value for the sample. Each assay used adifferent capture antibody. The first used a CD9 capture antibody, thesecond a CD81 capture antibody, and the third a CD63 antibody. The samecombination of detection antibodies was used for each assay, antibodiesfor CD9, CD81, and CD63. If the average value obtained for the threeassays was greater than 3000, the sample was categorized as havingoverexpressed exosomes (FIG. 79, Exosome).

Prostate Exosome Overexpression

The MFIs for a sample in two assays were averaged to determine a valuefor the sample. Each assay used a different capture antibody. The firstused a PCSA capture antibody and the second used a PSMA captureantibody. The same combination of detection antibodies was used for eachassay, antibodies for CD9, CD81, and CD63. If the average value obtainedfor the two assays was greater than 100, the sample was categorized ashaving prostate exosomes overexpressed (FIG. 79, Prostate).

Cancer Exosome Overexpression

Three different cancer bio-signatures were used to determine if cancerexosomes were overexpressed in a sample. The first, Cancer-1, used anEpCam capture antibody and detection antibodies for CD81, CD9, and CD63.The second, Cancer-2, used a CD9 capture antibody with detectionantibodies for EpCam and B7H3. If the MFI value of a sample for any twoof the three cancer bio-signatures was above a reference value, thesample was categorized as having overexpressed cancer (see FIG. 79,Cancer-1, Cancer-2, Cancer-3).

Reliability of Sample

Two quality control measures, QC-1 and QC-2, were determined for eachsample. If the sample met one of them; the sample was categorized asreliable.

For QC-1, the sum of all the MFIs of 7 assays was determined. Each ofthe 7 assays used detection antibodies for CD59 and PSMA. The captureantibody used for each assay was CD63, CD81, PCSA, PSMA, STEAP, B7H3,and EpCam. If the sum was greater than 4000, the sample was not reliableand not included.

For QC-2, the sum of all the MFIs of 5 assays was determined. Each ofthe 5 assays used detection antibodies for CD9, CD81 and CD63. Thecapture antibody used for each assay was PCSA, PSMA, STEAP, B7H3, andEpCam. If the sum was greater than 8000, the sample was not reliable andnot included.

The sensitivity and specificity for samples with BPH and without BPHsamples after a sample met the criteria as described herein, are shownin FIG. 79.

It will also be understood that the foregoing description is ofexemplary embodiments of the invention and that the invention is notlimited to the specific forms shown or described herein. Variousmodifications may be made in the design, arrangement, and type ofelements disclosed herein, as well as the steps of utilizing theinvention without departing from the scope of the invention as expressedin the appended claims.

1. A method of assessing a cancer in a subject comprising: identifying abio-signature of an exosome population isolated from said subject,comprising: a) determining the levels of: (i) CD9, CD63, CD81 biomarkerprotein or a combination thereof, of said exosome population; (ii) oneor more cell-specific biomarker of said exosome population; and (iii)one or more additional biomarker from said exosome population that isindicative of cancer; and b) comparing said levels of said biomarkersfrom said exosome population to a reference, wherein elevated levels ascompared to said reference indicate that said subject may be predisposedto or afflicted with cancer, thereby assessing said cancer.
 2. Themethod of claim 1, wherein the exosome population is isolated from abodily fluid from said subject.
 3. The method of claim 2, wherein saidbodily fluid is peripheral blood, serum, plasma, ascites, urine,cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid,aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolarlavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatoryfluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid,pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle,bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, orumbilical cord blood.
 4. The method of claim 1, wherein said exosomepopulation comprises vesicles having a diameter of about 10 nm to about800 nm.
 5. The method of claim 1, wherein said exosome populationcomprises vesicles having a diameter of about 30 nm to about 200 nm. 6.The method of claim 1, wherein said exosome population is isolated fromsaid biological sample with size exclusion chromatography, densitygradient centrifugation, differential centrifugation, nanomembraneultrafiltration, immunoabsorbent capture, affinity purification,microfluidic separation or a combination thereof.
 7. The method of claim1, wherein the cancer is prostate, lung, colon, breast, bladder,endometrial, liver, pancreatic, ovarian, esophageal or kidney cancer. 8.The method of claim 1, wherein the cancer is prostate cancer.
 9. Themethod of claim 1, wherein said determining comprises measuring anexpression level, presence, absence, mutation, truncation, insertion,modification, sequence variation or molecular association of saidproteins from said isolated exosome population.
 10. The method of claim2, wherein said bodily fluid is blood, urine, serum or plasma.
 11. Themethod of claim 1, wherein said characterizing comprises one or more ofdetermining an amount of exosomes, a temporal evaluation of a variationin exosome half-life, a temporal evaluation of circulating exosomehalf-life, a temporal evaluation of exosome metabolic half-life, ordetermining exosome activity.
 12. The method of claim 1, wherein saidcell-specific biomarker comprises PCSA and/or PSMA.
 13. The method ofclaim 1, wherein said bio-signature comprises one or more binding agentthat binds to at least one of said biomarkers.
 14. The method of claim13, wherein said one or more binding agent is an antigen, DNA molecule,RNA molecule, antibody, antibody fragment, aptamer, peptoid, zDNA,peptide nucleic acid (PNA), locked nucleic acids (LNA), lectin, peptide,dendrimer or chemical compound.
 15. The method of claim 1, wherein saidassessing comprises a diagnosis, prognosis, determination of drugefficacy, monitoring the status of said subject's response or resistanceto a treatment or selection of a treatment for said cancer.
 16. Themethod of claim 1, wherein said reference is derived from abio-signature identified in said subject over a time course.
 17. Themethod of claim 1, wherein said one or more additional biomarker is B7H3and/or EpCam protein.
 18. The method of claim 17, wherein saidcell-specific biomarker is PCSA and/or PSMA.
 19. The method of claim 1,wherein said cell-specific biomarker is PCSA.
 20. The method of claim 1,wherein said cell-specific biomarker is PSMA.
 21. The method of claim 8,wherein said one or more additional biomarker is miR-141.
 22. The methodof claim 8, wherein said one or more additional biomarker isTMPRSS2-ERG.
 23. The method of claim 17, wherein said one or moreadditional biomarker is miR-141 or TMPRSS2-ERG.
 24. The method of claim21, 22 or 23, wherein said biomarker is assessed by quantitative PCR,nucleic acid sequencing, hybridization assay or a combination thereof.25. A method of assessing a disease or disorder in a subject comprising:a) determining the amount of exosomes present in a sample isolated froma subject and b) comparing said amount to a reference, wherein elevatedlevels as compared to said reference indicate that said subject may bepredisposed to or afflicted with cancer, thereby assessing said cancer.26. The method of claim 25, wherein said disease or disorder isprostate, lung, colon, breast, bladder, endometrial, liver, pancreatic,ovarian, esophageal or kidney cancer.
 27. The method of claim 26,wherein said determining comprises detecting the presence or level ofCD9, CD63, CD81 or a combination thereof of said exosomes.
 28. Themethod of claim 25, wherein said determining comprises detecting thepresence or level of CD9 protein from said exosomes.